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University of Pittsburgh Schools of the Health Sciences

University of Pittsburgh Reports Best Gene Delivery to Date of Protein Missing in Duchenne Muscular Dystrophy

PITTSBURGH, November 27, 2000 — Using sophisticated techniques, University of Pittsburgh 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 research, conducted in animals and reported in the Nov. 28 issue of the Proceedings of the National Academy of Sciences, is remarkable on several fronts. The Pittsburgh team managed to whittle 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.

"Our research showed that we could introduce functional dystrophin into muscle tissue with AAV. 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, Ph.D., assistant professor in the department of molecular genetics and biochemistry at the University of Pittsburgh. "We were excited to see that mice treated with the mini-dystrophin gene did not show evidence of muscle breakdown many months after treatment, and even after the treated mice exercised, as was seen in untreated mice that lack dystrophin."

"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, Ph.D., chairman of the department of molecular biochemistry and genetics at the University of Pittsburgh 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. His design of a novel dystrophin mini-gene that both fits into this AAV and which corrects the dystrophin deficiency in the DMD mouse model is a significant achievement."

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

Considered the most common genetic ailment, Duchenne Muscular Dystrophy, or DMD, is an X-linked disorder that 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.

In boys lacking dystrophin, muscles function abnormally, at first enlarging, then degenerating so that fat and scar tissue take over. Boys with DMD lose muscle function throughout their bodies, until either the heart or the muscles that control breathing are compromised to the point where they no longer sustain life.

"This news moves us one step closer to a cure and for now provides real hope to further help these boys," said Ms. Pat Furlong, president of the Parent Project for Muscular Dystrophy Inc., which provided partial funding for this research.

Central to this research accomplishment was the work of Bing Wang, M.D., Ph.D., research associate, and Juan Li, M.D., senior research associate. Drs. 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, Dr. 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.

Dystrophin plays a critical role in maintaining the integrity of muscles located throughout the body. Within each muscle cell are bundles of actin and myosin filaments. Stacked on one another, the actin and myosin proteins move back and forth in a plane, thereby effecting muscle contraction. Dystrophin forms a bridge between the muscle cell membrane and each of these contractile bundles of actin and myosin. Like a spring, each dystrophin protein allows the actin and myosin to move smoothly and effectively, while they remain anchored to the cell’s interior.

By maintaining the muscle cells’ integrity, dystrophin also prevents muscle cells from becoming "leaky" to surrounding molecules. Muscle cells from DMD mice – and patients – show this increased "leakiness," which eventually contributes to their demise and replacement with fat and scar tissue.

Dr. Xiao’s group truncated the dystrophin genetic sequence that codes for a long series of repeat units in the middle of the dystrophin protein. Dr. Xiao’s mini-gene construct effectively shortened the "spring" by removing some of the repeats within it. Even with much fewer repeat units, the protein’s function was nonetheless remarkably preserved. The "spring" apparently remained flexible enough to perform its function. The mini-dystrophin gene also contained structures for this series of repeats to remain anchored effectively on one end to the muscle cell membrane and on the other end to an actin filament.

For many years, adeno-associated viruses were virtually ignored by researchers because they mistakenly thought these tiny microbes failed to infect non-dividing cells and because they could only carry a small genetic payload. Yet, scientists are now quite excited by the biological properties of AAV, according to Dr. Xiao.

The AAV vector is considered ideal for clinical gene therapy for many reasons, according to Dr. Xiao. The virus from which the AAV vector is produced does not cause any diseases and is disabled so that it cannot reproduce. The AAV vector is easily manufactured and stable. It can be targeted to specific tissues and produces long-lasting gene expression. Moreover, it does not provoke an immune response against cells that it enters. This type of immune response has been seen in gene therapies using other virus types.

In a next step, Dr. 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 at delivering the DMD mini-gene systemically, as opposed to performing localized injections.

In addition to the support from the Parent Project, this research also received grant support from the National Institute of Arthritis and Musculoskeletal and Skin Diseases.

The Parent Project is a national organization founded in 1996 and managed by parents whose children have Duchenne (du-shen) or Becker Muscular Dystrophy. The organization works in conjunction with the "Kids for Kids Project" to provide support, cutting-edge research, treatment and hope.

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