(From the September 2015 Issue of PediatricsOnline)
Researchers at Nationwide Children's Hospital have restored function in mouse models of human dysferlinopathies, a family of untreatable muscle disorders, using a new gene therapy technique.
Scientists believe that replacing the abnormal dysferlinprotein coding gene found in limb girdle muscular dystrophy type 2B, Miyoshi myopathy, distal anterior compartment myopathy and other members of the disease family could halt muscular degeneration, if not provide a cure.
But the gene, dysferlin cDNA, is too large to fit inside an adeno-associated virus (AAV), the vector that is best-suited to transport material into muscle cells.
To solve this mismatch, the research team separated the dysferlin gene into two overlapping sections, packaged them in separate AAVs and injected both into mouse models.
Inside muscle cells, "we hijack the cell's DNA repair mechanisms to make a whole gene," says Louise Rodino-Klapac, PhD, a principal investigator in the Center for Gene Therapy at the Research Institute at Nationwide Children's Hospital and the study leader.
Whether the dual vectors were injected into muscle or the bloodstream, periodic testing from one month to one year later showed that mouse models expressed high levels of dysferlin in their muscle. Periodic testing also showed the muscles repaired themselves as rapidly and were as strong as those in wild type mice, the researchers report in Annals of Clinical and Translational Neurology.
Dysferlinopathies strike between one in 100,000 and one in 200,000 people worldwide, usually during the second decade of life. Nearly one-third are wheelchair-dependent within 15 years.
The common factor among the diseases is the lack of the protein dysferlin, which is required to repair damaged muscle membranes. Without the protein, the damage leads to muscle fiber degeneration, inflammation, weakness, eventual replacement by fat and then fibrosis.
To restore dysferlin cDNA, Rodino-Klapac's team made cassettes containing the 5' and 3' portions of the gene, which include an overlap of about 25 percent of the genetic material. Each portion was packaged inside its own AAV of serotype Rh74 and injected into the muscle, into an artery near muscle or in the tail vein.
When the cassettes enter a cell nucleus, the AAV capsid, or protein coat, is stripped away, exposing the genetic material. The overlapping sequences signal to the cell's own DNA repair mechanisms that the pieces belong together and serve as a guide to recombine them into a functioning gene, explained Rodino-Klapac, who is also assistant professor in the Department of Pediatrics at The Ohio State University College of Medicine.
Each delivery method resulted in functioning genes.
"We were unsure the technique would work systemically because you need both pieces of the gene in each muscle cell," Rodino-Klapac says. "But, the results show the gene segments were dispersed and recombined in muscles throughout the whole body, which is what's needed to translate this into a meaningful therapy."
Large animal models treated with direct muscle injection also showed strong dysferlin expression in regular testing over six months. The large animal and mouse models showed no toxic effects from the treatment.
The researchers are applying for U.S. Food and Drug Administration approval to run clinical trials, which they believe may be the first using a two-vector approach.
Sondergaard PC, Griffin DA, Pozsgai ER, Johnson RW, Grose WE, Heller KN, Shontz KM, Montgomery CL, Liu J, Clark KR, Sahenk Z, Mendell JR, Rodino-Klapac LR. AAV. Dysferlin overlap vectors restore function in dysferlinopathy animal models. Annals of Clinical and Translational Neurology. 2015 Mar;2(3):256-70.