Mayopathy(Institute of Muscular Dystrophy & Research Center)

 Dystrophin-Deficient Dogs Benefit From Gene Therapy

University of Missouri.

Jan 15 2013.

MDA-supported investigators found that intramuscular injections of microdystrophin genes

 improved muscle health in dystrophin-deficient dogs, a response not previously seen in large

 animals or humans

MDA research grantee Dongsheng Duan and colleagues have found that dogs with a DMD-like disease can be successfully treated with microdystrophin gene therapy

Article Highlight:

• Researchers supported in part by MDA used highly miniaturized dystrophin (microdystrophin) genes encased in AAV9 delivery vehicles to treat six dystrophin-deficient dogs that had a disease mimicking human Duchenne muscular dystrophy (DMD).
• The gene injections were made directly into the dogs’ front leg muscles.
• Muscle fibers that received the gene therapy showed good dystrophin protein production, improvements in muscle appearance, and partial protection from the weakness that occurs in dystrophin-deficient muscles after repeated contractions.
• No specific immune responses were detected against the newly made microdystrophin protein or the vehicle used to deliver the new genes, although immune system T cells were seen in the treated muscle fibers.

For the first time, gene therapy using a highly miniaturized dystrophin gene resulted in significant improvement in muscle structure and function in dogs with a disorder mimicking human Duchenne muscular dystrophy.

The MDA-supported findings may help advance the development of microdystrophin gene therapy for DMD and the related disorder Becker muscular dystrophy (BMD), both of which result from a deficiency of the dystrophin protein.

MDA research grantee Dongsheng Duan, a professor in the Department of Molecular Microbiology and Immunology at the University of Missouri in Columbia, coordinated the research team, whose findings were published online Jan. 15, 2013, in the journal Molecular Therapy.

Microdystrophin gene therapy has been in development for more than a decade but so far has shown better results in dystrophin-deficient mice than in dystrophin-deficient dogs or humans with DMD.

This is the first time that significant muscle-related benefits have been seen with microdystrophin gene therapy in a large animal model of DMD.

Although the results were encouraging, the researchers note that the response of the dogs to microdystrophin gene therapy was not as robust as the response of dystrophin-deficient mice in previous experiments. They say further studies are needed to create an optimal microdystrophin gene, AAV delivery system and regimen of immunosuppressive drugs.

Immune response has been a challenge

MDA-supported investigators reported in late 2010 that four out of six boys with DMD who received microdystrophin gene therapy into a biceps muscle showed evidence that their immune systems rejected the newly synthesized dystrophin protein.

Previously reported studies of microdystrophin gene therapy in dystrophin-deficient dogs also have shown less than optimal results and some evidence of rejection of the therapy by the immune system.

It is widely believed that the dog model of DMD is more like the human disease than are mouse models. The canine immune system may also replicate the human immune system better than the mouse immune system does.

Improvements seen in muscle fiber structure and function

Six dystrophin-deficient dogs received injections into front leg muscles of microdystrophin genes, each encased in a delivery vehicle made from a modified type 9 adeno-associated virus (AAV9). Four of the dogs received a single gene therapy injection into one front leg muscle; two dogs received a single gene therapy injection into each front leg muscle.

All six dogs received temporary immunosuppression using two drugs, cyclosporine and myocophenolate mofetil (CellCept), intended to help them tolerate the gene transfer.

Two months after the gene injections, when treated muscle fibers were compared with untreated muscle fibers, the investigators saw:

• robust production of dystrophin from the microdystrophin genes (microdystrophin protein);
• restoration of a cluster of proteins at the muscle-fiber membrane that is disrupted when dystrophin is absent;
• much less calcification of muscle;
• substantially less scar tissue (fibrosis);
• less invasion of muscle tissue by inflammatory cells;
• more normal muscle fiber size; and
• significant protection of muscle fibers against the weakness that occurs with repeated contractions in dystrophin-deficient muscles.

The investigators saw no evidence of an immune response against either the newly made microdystrophin or the viral delivery vehicle. However, somewhat surprisingly, they saw an abundance of immune system T cells in the treated muscle fibers. The effects of the T cells and the reasons for their presence remain unclear.

What makes these experiments different

Commenting on the relative success of the current gene therapy experiments in dystrophin-deficient dogs compared to other DMD dog gene transfer experiments, the investigators note that

• the specific microdystrophin construct used in these experiments was different from that used in other experiments;
• the experimental design was different from that used in other studies;
• the gene delivery strategy was different; and
• the age of the dogs was different (older).

"In summary," the researchers say, "our results have cleared uncertainty on microdystrophin therapy arisen from other dog studies. However, compared with what was reported in the mouse model, the improvement we saw in dystrophic dogs remained suboptimal."