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Genetic surgery for muscular dystrophy in golden retrievers
By Haleh V. Samiei

Animal models of Duchenne muscular dystrophy (DMD) do not respond well to current approaches to gene therapy that use DNA injection, viral vectors or myoblast transplantation. The length of the dystrophin gene involved in DMD and immunological reaction to viral proteins create major barriers. In this study, Richard J. Bartlett and collaborators overcome these barriers by testing a new approach that attempts instead to repair the dystrophin gene in a canine model of DMD in golden retrievers (GRMD).

Like the mdx mouse model for DMD, GRMD in this research group's colony of dogs is caused by a single mutation in their dystrophin gene, which is exactly like the human gene. This mutation alters the tailoring of the RNA message from the gene, resulting in a deletion of part of the message and thus the production of a cut-off dystrophin protein. There are other colonies of dogs with GRMD that have other types of mutations, such as deletions and insertions in the dystrophin gene.

The human dystrophin gene has mutations and deletions similar to the dog gene in DMD patients. Bartlett, who now has a joint appointment by the University of Missouri and the National Institute of Arthritis and Musculoskeletal and Skin Diseases (NIAMS), was part of the group who originally localized the human DMD locus to the X chromosome and identified deletions in patients' genes. It is prevalent in young human males.

‘We take the existing gene and correct it’

Just like humans, golden retrievers with the mutant dystrophin gene have weak muscles and eventually die from the disease. The dogs usually die by six months of age, but if they survive that crisis period, they can go on to live up to 3 to 5 years. These dogs then die of cardiomyopathy. "The heart muscle is just as affected in both the patients and the dogs," says Bartlett. The difference is that dogs can continue to move around with their early muscle problems since they are quadrupeds, but affected boys lose their ability to stay upright and must use a wheelchair. The boys die due to atrophy of muscles leading to respiratory problems before they get to the cardiomyopathy stage.

In Bartlett's current publication in the June issue of Nature Biotechnology, he and his colleagues perform "genetic surgery" to repair a mutant dystrophin gene in the muscles of a golden retriever with GRMD. "We take the existing gene and correct it," says Bartlett. This is all without introducing extraneous genes from any other organisms.

The researchers perform this microsurgery by designing and synthesizing a five-base DNA segment of the normal dystrophin gene that can enter muscle cells and pair up with the mutant gene right around the single base change. Attached to both ends of this DNA segment are very short stretches of RNA and DNA that allow the chimeric oligonucleotide molecule to pull apart the strands of DNA in the chromosome and adhere tightly to the surrounding DNA. The mismatch at the site of the mutation flags down the cell's DNA repair machinery and coaxes it to replace the chromosomal DNA using the available template, which happens to be the correct sequence of bases in the oligonucleotide.

The laboratory of Eric B. Kmiec of the University of Delaware originally invented this type of targeted gene repair. A few other researchers have used the technique for the treatment of sickle cell anemia and Criglar Najjar disease, a rare genetic defect causing jaundice and irreversible brain damage in the severe form. Just last month, Thomas A. Rando of Stanford University School of Medicine in Palo Alto, California, reported the successful use of the gene repair technique to correct the mouse dystrophin gene.

Bartlett and colleagues injected their chimeric oligonucleotide into affected muscles of a six-week-old male dog. Using molecular methods, they were able to demonstrate from muscle biopsy and necropsy samples that the dystrophin gene had been repaired, producing correct RNA messages and complete dystrophin proteins for up to eleven months. The researchers could also identify pockets of healthy muscle tissue.

Although this is the most promising treatment for DMD so far, many problems still need to be resolved before it can help people. "I firmly believe that we've got to get this working in the model first," says Bartlett, "before we attempt anything in the patients."

One major problem is delivering enough of the oligonucleotide to all affected muscles. "One experiment someday I would love to try with these dogs is to put a pump into the muscle and just constantly diffuse this chimera into the muscle and see if we can just jack the level up," says Bartlett. He and his colleagues are also investigating the possibility of delivering the repair oligonucleotide to mdx mouse muscles through electroporation, a mild electric shock that would allow absorption of the oligonucleotides through the skin.

The diversity of the human dystrophin mutations creates another problem. "With each new patient, it is a new mutation," says Bartlett. In order to treat each patient, the exact molecular nature of the mutation would have to be defined first. Then you would have to custom-design a specific oligonucleotide to correct that patient's mutation, says Bartlett. This would all take a tremendous amount of time and expense.

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Bartlett, R.J. et al. In vivo targeted repair of a point mutation in the canine dystrophin gene by a chimeric RNA/DNA oligonucleotide. Nat Biotechnol 18, 615-622 (June 2000).

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