Gene replacement, enhanced genetic testing, clinical trial s show promising future
RPE65 gene replacement therapy trials for Leber's congenital amaurosis (LCA) in both dogs and humans continue to appear promising.
"We're excited about that, not just because of this disease, but because of the way it seems to be leading the way toward gene replacement therapy for a lot of other diseases," said Dr Stone, professor of ophthalmology, Howard Hughes Medical Institute Investigator, and holder of the Seamans-Hauser Chair in molecular ophthalmology at the University of Iowa, Iowa City.
A third important development is advanced technology, specifically a combination of allele-specific testing and next-generation deep sequencing that is much faster and more accurate than older methods.
Dramatic improvements in genetic testing over the past few years and a wider range of clinical trials are hopeful signs.
"These things are a tremendous opportunity for those of us who see patients to reverse that negative message to families that there's nothing we can do, and to give our patients better diagnoses and better counselling," Dr Stone said. "They also contribute to the development of treatments, which depend on a correct molecular diagnosis."
He stressed, however, that it is important to realize that the vast majority of genetic variations in a person's DNA are not disease-causing. Each individual has millions of non-disease-causing variations, which will be found during genetic screening. Individuals who perform testing and communicate results must be well informed about which mutations are harmless and which could be associated with inherited retinal dystrophies.
Dr Stone also summarized the latest results from the LCA gene therapy trial at the University of Pennsylvania, which were recently published in The Lancet. All 12 patients in the trial have had their sight partially restored after a single injection of the therapeutic agent, and there is a trend for the younger patients to have the most improvement in vision. No severe adverse events have been reported in any patients, some of whom have now been followed for 2 years.
"This success will lead to enrolement of even younger patients, for whom there is a better chance of restoring good vision," he said.
Project 3000 ( http://www.project3000.org/) is a nationwide initiative to genotype every patient in the United States with LCA. Researchers have estimated that there are about 3700 people in this country with LCA, of whom 8%, about 300, have the form associated with RPE65. These are individuals who are, as Dr Stone explained it, spread across all ages. There are about 42 cases per decade, which equates to 84 patients under the age of 20 with retinal pigment epithelium-specific microsomal protein mutations (RPE65-LCA).
Dr Stone asked clinicians to support the project by helping identify patients of any age with LCA, adding that philanthropic funding is available to help offset the costs of genetic testing for individuals who do not have insurance.
He also described two new molecular technologies. From 1990 to 2005, the mainstay of mutation detection was a combination of single-strand conformation polymorphism (SSCP) analysis and DNA sequencing. Two overlapping strategies have evolved to overtake this approach, allele-specific testing and next-generation sequencing. Allele-specific testing is a rapid, inexpensive way of finding a mutation already known to cause disease using a nanotechnology tool from Fluidigm that can mix samples from 48 patients with 48 known disease-causing, allele-specific test reagents during a 3-hour polymerase chain reaction.
"This tool has 60-fold greater throughput with a given amount of lab space and personnel compared [with] SSCP, which we thought was the gold standard, and it's one-quarter the cost per genotype than the best thing we had before," Dr Stone said.
The disadvantage of this tool, which it shares with other allele-specific products, is that it does not detect every mutation. That is, it will not detect disease-causing variations that have not yet been discovered and those that are not part of the assay, he added.
Next-generation deep sequencing was adapted from full-genome sequencing projects. Dr Stone's lab uses a Roche-454 Sequencing System in which a DNA molecule from a patient is bound to a tiny bead, which is placed in a well on a glass plate that holds about 1 million of these wells. As reagents are poured across the plate, certain wells produce chemiluminescent signals that are captured by a camera. The sequencing technique can complete 1.25 million base pair sequences in an 8-hour run, resulting in 250-fold greater throughput than with conventional sequencing at one-twentieth of the cost. Careful follow-up is required to interpret the results correctly, said Dr Stone.