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doi: 10.1097/01.NT.0000335574.79492.d0
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Investigators used an adeno-associated virus to deliver the gene for alpha-sarcoglycan to a knockout mice model for limb girdle muscular dystrophy; there were none of the toxicity issues that hampered an earlier animal study of the technique.

Having successfully overcome safety concerns in animal models, a team of researchers has launched the first human trial using gene therapy to treat a form of limb girdle muscular dystrophy (LGMD2D) refractory to any treatment.

Jerry R. Mendell, MD, director of the Center for Gene Therapy at Nationwide Children's Hospital in Columbus, OH, and colleagues, used an adeno-associated virus (AAV) to deliver the gene for alpha-sarcoglycan (alpha-SG), a muscle protein, to knockout mice; there were none of the toxicity issues that hampered an earlier animal study of the technique.

LGMD2D is caused by a deficiency of alpha-SG. The results, reported in the July 22 Neurology, prompted investigators to submit an investigational new drug (IND) application to the FDA. Approval by the FDA enabled the investigators to begin testing the technique in their first patient in April. A second patient was scheduled to begin treatment in late June. He told Neurology Today in a telephone interview that gene transfer in the first patient went well without adverse effects. “This is encouraging and our intent is to move forward as soon as possible with vascular gene delivery so that multiple muscles can be reached,” he said.

Limb girdle muscular dystrophies encompass at least 19 different types, each caused by a specific protein defect. Faulty genes are responsible for the abnormal proteins that lead to improperly working muscle fibers and consequent muscle weakness, which is progressive and eventually leads to severe disability.

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In the mouse studies, the researchers injected the human alpha-SG gene directly into the anterior leg muscle of 4- to 5-week-old alpha-SG knockout mice using AAV type 1.

The gene was placed under the control of either the ubiquitously expressed cytomegalovirus (CMV) promoter or muscle specific promoters, including desmin, muscle creatine kinase (MCK), and the modified truncated form of MCK (tMCK), Low (3X109 vg) and high (3 X1010 vg) doses of AAV1 were used to transfer the human alpha-SG.

Prior research has shown that rAAV-mediated gene transfer of alpha-SG expression is either uncontrolled and therefore transient or cytotoxic when the ubiquitous cytomegalovirus promoter is used to drive expression. Muscle-specific promoters, including those used by the researchers, can reduce or avert inappropriate gene expression in surrounding non-target cells by controlling and restricting expression. In addition, limiting expression to the target site also reduces or prevents humoral or cell-mediated immune responses.

In the current study, there was sustained gene expression at six and 12 weeks, irrespective of the promoters used.

“Our data demonstrate robust and sustained adeno-associated virus type 1 alpha sarcoglycan gene expression under control of muscle creatine kinase promoters, without evidence of toxicity,” said Dr. Mendell. “These findings support the use of gene therapy as a possible treatment for limb girdle muscular dystrophy type 2D.”

Although muscle toxicity and rapidly attenuating gene expression from overexpression of the alpha-SG gene have been concerns in the past, the new data show that the risk can be eliminated using MCK, tMCK, or a synthetic muscle-specific promoter (C5-12), according to Dr. Mendell. Immunofluorescence data demonstrated that MCK and tMCK promoters enabled robust and long-term gene expression for 12 weeks after the vector injection. Expression under CMV trended downward at the 12-week time point.

As encouraging as the findings are, Dr. Mendell said that it is too soon to tell whether the technique will work as well in humans.



“Extrapolating findings from mice to humans requires an air of cautious expectation with regard to safety and efficacy,” he said.

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In a 2002 study in Human Gene Therapy by Devin Dressman, PhD, and colleagues at the Children's National Medical Center in Washington, DC, alpha-SG knockout mice were given AAV type 2, CMV, and alpha-SG at a dose comparable to the present study and in the range that would have been used in a human trial. But there were fewer than 4 percent of alpha-SG positive fibers in most treated muscles six weeks after gene transfer. The small number of transduced fibers indicated a significant drop off in gene expression between weeks four and six and would have been insufficient to warrant a clinical trial, according to Dr. Mendell.

“Possible explanations for the observed differences between our findings and the earlier report include the choice of serotype, purification method for the AAV, or differences in vector design,” he said. “The serotype is an obvious difference. Multiple studies have documented more favorable gene expression in muscle using AAV1 versus AAV2 at comparable doses. In our studies we documented more than two-fold alpha-SG overexpression without cytotoxicity, predicting a favorable clinical response.”

Introducing the treatment to the clinic requires “a stepwise approach,” noted Dr. Mendell. First, the local muscle injection must prove safe, with sustained gene expression. Because the new study showed that the technique accomplished this in mice, he continued, it lays the foundation for testing a vascular approach, with the objective of reaching multiple muscles of a limb and potentially clinically meaningful results for patients.

“We've gotten good results in mice and nonhuman primates with vascular gene delivery,” he said. “At some future point, our strategy is to go back to the FDA with results from clinical intramuscular gene transfer hopefully showing no adverse effects. Then, we could do a vascular delivery trial if all goes well. I think a treatment could be available in five years, but that's a conservative estimate — I think we can get there faster.”

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Although the results are encouraging, the discrepancy between the new findings and the toxicity reported in the earlier study argues for caution, said R. Jude Samulski, PhD, director of the Gene Therapy Center at the University of North Carolina Medical Center at Chapel Hill.

“While the data in this study support expression of the alpha-SG gene, the controversy with the earlier study still raises concerns,” he told Neurology Today by telephone. “It's important to know why one worked and the other didn't. Without knowing, it's hard to know how to go forward. Both are respectable research groups, so I think that their different findings mean we need to proceed carefully.” •

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• Rodino-Klapac LR, Lee JS, Mendell JR, et al. Lack of toxicity of alpha-sarcoglycan overexpression supports clinical gene transfer trial in LGMD2D. Neurology 2008;71:240–247.
    • Dressman D, Araishi K, Imamura M, et al. Delivery of alpha- and beta-sarcoglycan by recombinant adeno-associated virus: efficient rescue of muscle, but differential toxicity. Hum Gene Ther 2002;13:1631–1646.
      • Gregorevic P, Blankinship MJ, Allen JM, et al. Systemic delivery of genes to striated muscles using adeno-associated viral vectors. Nat Med 2004;10:828–834.
        ©2008 American Academy of Neurology