|University of Western Australia, Perth|
Steve Wilton likes to call his gene modification strategy a “genetic Band-Aid.” Like Tom Rando, Wilton and colleagues are using oligonucleotides — short pieces of genetic material — to change the way cells read genetic instructions.
But there are some key differences between Rando’s gene-correction strategy and Wilton’s Band-Aid technique. Wilton’s plan involves blocking the part of the “rough draft” genetic blueprint (pre-mRNA) that contains a flaw.
His oligonucleotides are called antisense, because they stick to and block parts of the RNA that the cell would ordinarily read (make sense of).
At this stage, the cell normally cuts out the parts of the RNA — the introns — that won’t be part of the final message, leaving only the parts of the final RNA message — the exons.
But an error in the gene that makes it into the pre-mRNA can cause the cell to cut and splice in the wrong place, so that the final instructions contain material that should have been edited out, or don’t contain material that should have been left in (a splice site error).
In the early 1990s, Wilton had completed his doctoral training at the University of Adelaide in Australia and was working for a small biotechnology company in Perth, making molecular biology compounds that other researchers would use. “I was getting very sick of that, and I wanted to get back to research,” he says, and he began working after hours at the Australian Neuromuscular Research Institute, part of the University of Western Australia.
At the institute, Wilton worked with molecular biologist Nigel Laing, who had been at Duke University in North Carolina with MDA grantee Allen Roses. “That’s how I got involved in muscular dystrophy,” Wilton recalls.
The gene for dystrophin had recently been found, and other genes for neuromuscular conditions were being identified at a rapid pace. While developing genetic testing for DMD and other diseases, Wilton became fascinated by a recently discovered phenomenon known as revertant fibers, muscle cells found in boys with DMD that mysteriously begin making dystrophin despite genetic mutations that should keep them from doing so.
Antisense makes sense
Wilton had an idea that the revertant fibers might occur when a glitch in the cell’s gene-reading machinery allowed it to “skip” a genetic error and continue making the protein from instructions on the far side of it.
“I had a limited imagination, I suppose,” he says, “because I couldn’t see any other way that could happen. Gene deletions are a common type of defect in the dystrophin gene, so it seemed logical that a second mutation could overcome the first one, a case of two wrongs making a right.”
Wilton’s imagination was correct. Exon skipping, as the phenomenon came to be called, was the mechanism by which dystrophin-containing fibers sometimes occurred despite mutations in the dystrophin gene.
But it wasn’t until October 1996, while listening to a lecture by Richard Kole of the University of North Carolina at a gene therapy conference in Lake Tahoe, Nev., that Wilton began to think about how to make exon skipping a treatment for DMD.
Kole was talking about using antisense constructions to block splice site mutations in the beta-globin gene, which underlies the blood disease thalassemia. Wilton recalls that, as his thinking strayed to the implications for dystrophin gene alteration, “it was like being hit by a brick.”
If splice site mutations could be blocked by antisense oligonucleotides, he thought, why not try blocking normal splice sites to keep error-containing exons from being included in the final mRNA?
Wilton and Kole struck up a conversation, and Kole agreed to send Wilton some antisense constructs. “A month after that, we had exon skipping working in some cultured cells.”
These days, having obtained equipment to synthesize antisense oligonucleotides quickly and relatively inexpensively in his own lab, Wilton says he’ll try “blocking anything” that looks like it will help someone with a dystrophin mutation.
“We have some exons where the donor splice site — that’s the one at the back of the exon — works really well. We’ve got other targets where it’s the front exon splice site — the acceptor — and sometimes it’s somewhere in the middle. Sometimes we get all three working. You can’t say there’s a best target as far as we have been able to tell.”
In 2003, he and his colleagues showed that a particular antisense oligonucleotide can overcome a premature stop codon mutation in the dystrophin gene in mdx mice and allow the animals to produce normal levels of dystrophin in a large number of muscle fibers.
Wilton now has the ear of a major pharmaceutical company that’s interested in applying exon skipping to DMD. “Honestly,” he says, “the last couple of years have been unbelievable, and with the support of industry and MDA, we’ll soon see if exon skipping is going to be a viable treatment.”