Stephen Wilton, an MDA grantee, and colleagues at the University of Western Australia in Perth, with scientists at AVI BioPharma in Corvallis, Ore., report significant progress in two sets of experiments that make use of the technique known as exon skipping.
This strategy coaxes cells to produce some functional dystrophin — the protein missing or inactive in Duchenne muscular dystrophy (DMD) — despite the presence of a genetic mutation, by skipping over exons (regions of genetic information) in the part of the gene with the defect. The skipping is accomplished by delivering antisense oligonucleotides (AOs) to cells.
On May 25, Graham McClorey and colleagues reported online in Gene Therapy that they’d achieved success in causing muscle cells taken from dogs with a disorder similar to human DMD to produce functional dystrophin.
They used AOs that carried a tag to enhance cellular delivery. These morpholino AOs, which appear to be more stable and taken up by cells more efficiently than other AO chemistries, block parts of the canine dystrophin gene transcript near the site of a serious genetic mutation.
“This allows the treated canine cells to ‘spit out’ the bad part of the defective dystrophin gene transcript to restore the reading frame [the way the cell reads the instructions] and allow synthesis of a functional gene product,” Wilton said.
The canine mutation mimics some that human patients have.
The exon skipping treatment allowed production of nearly full-length dystrophin, with no apparent adverse effects on the cells, the researchers say.
The induction of dystrophin in the [dog] model offers the potential for further testing of AO delivery regimens in a larger animal model of DMD, in preparation for application in human clinical trials,” the investigators write.
Abbie Fall and colleagues, in a study published online May 24 in Genetic Vaccines and Therapy, applied the strategy to a wider range of genetic mutations than had previously been targeted.
|Exon Skipping: As a cell prepares the final version of instructions for making a protein, it removes excess material and leaves only the exons, the parts that will form the final protein recipe. Laboratory-designed antisense compounds can make a cell eliminate a specific exon along with the other unwanted material.|
Using cocktails of morpholino AOs, these researchers induced cells to skip a large section of the dystrophin gene in mice carrying a “stop signal” mutation in exon 23. Previous successes in these mice only eliminated exon 23, splicing together exons 22 and 24, but this time, the researchers induced skipping of exons 19 to 25.
They found detectable, apparently functional dystrophin eight weeks after a single intramuscular injection into 11-day-old and 16-week-old mice.
In the younger mice, dystrophin appeared only in the area around the injection site but was strong and consistent. In the older mice, dystrophin was more widely distributed, but was patchy.
Analysis of dystrophin gene defects that give rise to mild cases of Becker muscular dystrophy, a less severe disease than DMD, has shown that loss of up to 66 percent of the dystrophin gene can still produce a fairly functional dystrophin protein and lessen disease severity, the investigators note.
The researchers say targeting multiple exons appears to be a “feasible strategy,” and that they could theoretically target more than 75 percent of dystrophin mutations that cause human DMD with a relatively small set of AO cocktails.
A clinical trial to test exon skipping with antisense in nine boys with DMD is scheduled to begin recruiting by the end of the year. The trial is under the auspices of the Imperial College of London and the Department of Health of the United Kingdom.
Investigators plan to inject varied doses of antisense constructs into a foot muscle and evaluate the safety of the pro-cedure and the amount and longevity of any dystrophin produced.
For updated information, see www.clinicaltrials.gov; enter “Duchenne muscular dystrophy” in the search box and scroll to the antisense trial.