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In Gene Therapy Trial for DMD, No Evidence of Sustained Gene Expression But a T-Cell Response is Reported

Talan, Jamie

doi: 10.1097/01.NT.0000390834.89099.d5
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A small gene therapy trial with six boys with DMD found that that the transfer of a transgene for dystrophin was safe, but investigators found that two boys had had a T-cell response to dystrophin even before the therapy was administered.

Gene therapy initiated in six boys with Duchenne muscular dystrophy (DMD) showed no evidence of sustained expression of a functional dystrophin gene, according to a study published Oct. 7 in the New England Journal of Medicine. But the investigators reported that they identified a surprising T-cell response that they say could contribute to the lack of gene expression.

DMD is caused by mutations in the gene encoding dystrophin, but efforts to use gene therapy to replace the defective gene in clinical trials have been hampered by the fact that the dystrophin gene — at 79 exons — is too large to fit inside a recombinant adeno-associated vector (rAAV).

In the current phase 1 safety study, which was conducted between 2006-2009, the investigators wanted to determine if a functional miniaturized dystrophin transgene, effective in mouse models of DMD, was expressed in muscle and whether it would elicit T-cell immunity in DMD patients. Jerry Mendell, MD, director of the Center for Gene Therapy at The Research Institute at Nationwide Children's Hospital and professor in neurology, pediatrics, pathology, physiology and cell biology at the Ohio State University, led the study.



In blood samples taken before the introduction of the gene therapy vector investigators found that two of the six patients had a T-cell response to dystrophin. The investigators attributed this T-cell response to the expression of revertant dystrophin, a term used to describe a small percentage of muscle cells (about one percent) that produce functional dystophin in the majority of DMD patients. These muscle cells have self-correcting genes, the investigators noted, but clearly they do not stop the muscle wasting.

Four hours before the administration of the gene therapy all of the six children received intraveneous methylprednisone to reduce the risk of local inflammation. The investigators then injected one bicep muscle with an rAAV vector that included a modified capsid of serotype 2 and a functional mini-dystrophin transgene — which was 40 percent of the 11-Kb coding sequence of the human dystrophin gene, according to Christopher Walker, PhD, the Wilby S. Cowan Endowed Chair in Pediatric Research in the College of Medicine and Public Health at Ohio State University and an investigator in the study. Three boys (patients 1-3) received low-dose vector and the other three (patients 4-6) received high-dose vector.

The investigators assessed gene transfer by examining biopsy specimens of vector-injected biceps muscles and untreated biceps muscles on day 42 (in patients 1,3,4, and 6) or day 90 (in patients 2 and 5). There was little, if any, dystrophin being expressed in the treated muscle. But the functional dystrophin transgene was well tolerated by the boys, Dr. Walker said, and there were no unusual side effects.

The investigators tested the patients' immune response by looking at the response of the white blood cells in culture. Mini-dystrophin-specific T cells were not detected at any time in two of the patients who had received low doses of the vector. But a third patient who also received a lower dose of the gene therapy had a robust T-cell activity against dystrophin (in a test tube) just two weeks following the therapy.

By contrast, T-cell responses were substantially delayed in two patients — patients 5 and 6 — who had received higher doses. The T-cell response in one of the patients who had shown immune activity prior to the treatment showed an intermittent immune response throughout the two-year follow-up period.

“In other studies in humans, transgene expression has not been associated with a cellular immune response against the therapeutic protein,” the authors wrote. “Detection of spontaneously primed T cells in the blood of patients 2 and 4 before they were treated with vector was unexpected. The rapid T-cell response that we observed after therapeutic gene transfer in patient 2 is consistent with a memory response boosted by the mini-dystrophin protein. Why the response was not boosted in patient 4 is unknown, but mini-dystrophin expression may have been inadequate to provoke a recall response.”

White blood cells naturally invade the muscle of patients with Duchenne muscular dystrophy, Dr. Walker commented. If dystrophin protein contributes to this inflammatory process “in some patients, it may complicate our efforts to restore muscle function,” he said. Still, he added, the small phase 1 study may have helped scientists learn something important about DMD. “Our finding suggests that there is a naturally occurring T-cell immunity that may target these self-correcting muscle cells. It is similar to an auto-immune response and it may contribute to muscle damage.”

The potential for a pre-existing cellular immune response to dystrophin that is generated naturally as part of the disease process should be considered in the design of future gene therapy trials for DMD, said Dr. Walker.



Muscle biopsies to study the immune response are needed to get a better picture of what is going on at the actual injection site. Those studies are in the planning stages.

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“The paper in question was a proof of principle,” said Louis Kunkel, PhD, director of the Genomics Program at Children's Hospital in Boston; professor of pediatrics and genetics at Harvard Medical School, and the scientist who identified the genetic defect in Duchenne muscular dystrophy in 1986.

Dr. Kunkel, who was not involved in the study, said that the findings should have “ramifications for all therapies that will attempt to restore dystrophin expression… The surprise was that two of the patients had the T-cell response todystrophin prior to the introduction of the vector.”

The immunity issues are being addressed in animal studies. Rainer Storb, MD, director of the Transplantation and Biology Program at the Fred Hutchinson Cancer Research Center and professor of medicine at the University of Washington, has conducted extensive work on canine models of DMD. He said that the field is aware of the problems of immunity to both the vector (as other animal and clinical studies have shown) and transgenes.

In a 2007 paper in Molecular Therapy, investigators described the problem of autoimmunity to vectors and transgenes and how they solved it in animal models of muscular dystrophy. They administered five doses of a commonly used biological, immunosuppressive agent, anti-thymocyte globulin, and six months of a drug combination that's also commonly used in marrow and organ transplantation, cyclosporin and mycophenolate mofetil. The drug doses were adjusted so that side effects were minimal. And the cells were not destroyed by an immune system reaction, he said.



“Extended follow-up of the animals has gone on for more than two years and it shows continued transgene expression,” said Dr. Storb. “So, I don't think gene therapy for muscular dystrophy is dead.”

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Mendell JR, Campbell K, Walker CM, et al. Dystrophin immunity in Duchenne's muscular dystrophy. N Engl J Med 2010;363:1429-1437.
    Wang Z, Kuhr CS, Storb R, et al. Sustained AAV-mediated dystrophin expression in a canine model of Duchenne muscular dystrophy with a brief course of immunosuppression. Mol Ther 2007; 15:1160-1166.
      ©2010 American Academy of Neurology