BioWorld International Correspondent

LONDON - Animal tests of a novel type of genetic therapy for muscular dystrophy showed highly encouraging results, according to a recent report. Clinical trials in people with muscular dystrophy could follow if researchers can find a way to deliver the therapy via the bloodstream.

The therapy aims to induce muscle cells to make functional dystrophin, the protein that is normally absent or faulty in people with muscular dystrophy. It relies on delivering a small piece of modified RNA to the nuclei of muscle cells.

Tests have so far been carried out only on a mouse model of muscular dystrophy, and the researchers who carried out the work warn that many more investigations need to be completed before the treatment can be tried on people. They are trying to find a mode of delivery that will carry the therapy to all muscles in the body via the bloodstream, rather than needing to be given by injection into the muscles.

Terence Partridge, head of the Muscle Cell Biology Group at the Medical Research Council's Clinical Science Centre at Hammersmith Hospital in London, told BioWorld International: "With a single injection into a muscle, we were able to convert about 20 percent of its component muscle fibers to producing normal amounts of dystrophin. This was a surprise because things hardly ever work with that level of efficiency."

A paper describing the experiments is published in the July 6, 2003, advance online Nature Medicine. Its title is "Functional amounts of dystrophin produced by skipping the mutated exon in the mdx dystrophic mouse."

When making normal dystrophin, the cell first manufactures messenger RNA along the entire length of the dystrophin gene. It then has to splice together the 79 exons of the RNA (which comprise about 1 percent of the entire gene) to make a template along which the ribosome runs as it reads the genetic code and adds one amino acid after another to the growing protein.

In humans, Duchenne muscular dystrophy is caused by mutations in the gene encoding dystrophin that result in the cell producing a truncated, nonfunctional protein. The same is true for the mouse model for the disease, called the mdx mouse.

The idea for the therapeutic approach described in Nature Medicine arose because Partridge, together with co-author Qi Long Lu at Hammersmith Hospital, had been intrigued for some time about the appearance of so-called "revertant fibers" in the muscles of both boys with Duchenne muscular dystrophy and the mdx mouse. Studies had shown that this phenomenon was not caused by a new mutation, but that the cells somehow managed to bypass the causative mutation, and continue to make the rest of the protein.

Together with Stephen Wilton and colleagues of the Queen Elizabeth II Medical Centre University of Western Australia in Nedlands, Partridge and Lu decided to investigate ways of getting cells to do this deliberately. Wilton had come up with a strategy of putting short antisense oligoribonucleotides into the cells, to force the gene-translation machinery to "skip" around the area containing the mutation, while Lu had been pursuing ways of improving the efficiency of getting genes into cells for gene therapy.

Their plan was to introduce a small "patch" of RNA to the nucleus of the muscle cell that would stick to the corresponding and specific piece of RNA that is copied from the DNA template in the dystrophin gene. The patch interferes with the RNA splicing mechanism, causing it to skip around the targeted exon. As a result, the cellular machinery that translates the messenger RNA into protein is not confronted by the mutation that normally brings it to a halt, and continues reading the genetic code on the other side of the mutation.

Partridge and his collaborators injected a mixture of a non-ionic block polymer called F127 (to facilitate delivery to the cells) and the RNA patch - an antisense oligoribonucleotide called 2OmeAO - into the tibialis anterior muscle of 4-week-old mdx mice. Within two weeks, the team was able to detect large numbers of fibers expressing normal levels of dystrophin.

Partridge said it is not clear exactly how the therapy works, but it appears to force the cell to read the message on either side of the mutated exon, and then splice together the unaffected exons, allowing the manufacture of a partially functional protein.

He added that the therapy has the advantage that, because it uses the patient's own gene, it is unlikely to trigger immunological problems. Furthermore, because the effect of the therapy is not permanent - unlike some kinds of gene therapy that have been proposed - the type of antisense nucleic acid given can be changed if necessary.

Partridge and his collaborators are investigating ways of altering the concentrations and amounts of F127 and 2OMeAO to find out if the nucleic acid can be delivered via the bloodstream to all muscles in sufficient quantities. They also want to find out if other substances related to F127 might be any more effective as a delivery vehicle. Work is also continuing on devising other antisense "patches" that are targeted on other commonly mutated exons in the dystrophin gene.