Split-Gene Delivery Strategy Explored
In Mice With Duchenne Dystrophy

MDA grantee Dongsheng Duan at the University of Missouri in Columbia led a research team in using a new strategy to deliver dystrophin genes to mice with Duchenne muscular dystrophy (DMD). The team included Jeffrey Chamberlain at the University of Washington-Seattle, who heads a muscular dystrophy center of excellence supported by MDA and the National Institutes of Health.

Dystrophin, the protein missing in DMD, is made from an extremely large gene, which has to be downsized to fit into a viral shell that can deliver it to muscles. Other MDA-supported groups, including one led by Chamberlain, are pursuing gene therapy with highly miniaturized (“micro”) dystrophin genes. Somewhat shortened, but still functional, dystrophin protein molecules can be made from these microgenes, but it’s still uncertain how much impact these microdystrophins will have on Duchenne dystrophy.

Now, Duan’s team, which published its results online Oct. 9 in Nature Biotechnology, has found that longer (“mini” instead of “micro”) dystrophin protein molecules can be made if the dystrophin gene is split into two specially engineered pieces, designed to rejoin after they emerge from their viral shells, with each piece placed in a separate viral transporter.

The researchers inserted each split-gene piece into a modified type 6 adeno-associated virus (AAV6) shell and injected the loaded shells into leg muscles of dystrophin-deficient, DMD-affected mice.

In 2-month-old mice, minidystrophin molecules showed up in more than half of the muscle fibers in the injected areas, where they protected fibers from injury and increased their force-generating capacity. In 1-year-old mice, an average of 40 percent of the fibers produced minidystrophin, but resistance to injury wasn’t as good as in the 2-month-olds.

The investigators say the split-gene strategy may have important implications for delivering not only dystrophin but other genes that are too large to fit into an AAV shell. They also say the results suggest that gene transfer may be more effective in younger patients, although it might still be somewhat effective in older ones.