By Dean Haycock

Special to BioWorld Today

Typically, the first bad sign comes to children at night. In the years following the onset of night blindness, peripheral vision slowly fades. In time, total darkness falls. Around the world, this sequence of creeping blindness affects one in every 3,000 to 4,000 individuals. The cause is retinitis pigmentosa (RP).

"Retinitis pigmentosa is a devastating disease," said Paul Sieving, director of the Center for Retinal and Macular Degeneration at the University of Michigan at Ann Arbor. Patients, he added, are desperate for a treatment.

Different mutations are responsible for the degeneration of the retina that characterizes RP. In most cases, RP can be traced to inheritance of a single, non-sex-related gene, that is, in an autosomal dominant pattern. In a quarter of these individuals, the RP mutation affects the gene that encodes rhodopsin, a molecule that detects light in the retina.

"Nearly any mutation in the [rhodopsin] molecule leads to death of the light-sensitive photoreceptor cell," Sieving said. "Any amount of rescue would be beneficial."

As devastating as it is, RP has some features that might be exploited. For instance, it progresses slowly, providing a potential window of opportunity for gene therapy treatments. Also, nature's reliance on redundancy has provided humans with more photoreceptor cells than they need in order to see. So, it may not be necessary to save all rhodopsin-containing cells in the retina threatened by RP, only enough to spare vision. Finally, because the disease is produced by just one version, instead of two versions, of the mutated rhodopsin gene in each cell, the non-mutated gene may be able to exert positive effects if the mutated gene can be neutralized.

Alfred Lewin and William Hauswirth, professors of molecular genetics and microbiology at the University of Florida in Gainesville, along with their graduate student Kimberly Drenser and their colleagues, are already exploiting these features of RP in hopes of developing the first effective treatment for the disease. A report in the August issue of Nature Medicine, "Ribozyme rescue of photoreceptor cells in a transgenic rat model of autosomal dominant retinitis pigmentosa," describes their approach.

The authors recognized three crucial parts of the research puzzle they would need to assemble to demonstrate the feasibility of their strategy. They needed an animal model of RP, a weapon to deactivate the mutated gene and a way to deliver the weapon.

Matthew LaVail of the University of California School of Medicine, in San Francisco, and John Flannery of the University of California at Berkeley provided a transgenic line of RP rats that have the same rhodopsin gene mutation found in the human disease. The weapon came in the form of a type of RNA called ribozymes. These enzyme-like small molecules bind to and cut up messenger RNA (mRNA) targets. For this study, ribozymes that destroyed messenger RNA carrying the code for mutated, but not normal, rhodopsin were prepared.

Results Represent Pair Of 'Firsts'

In the US, 12 percent of patients with autosomal dominant RP carry a mutation in which a histidine amino acid is substituted for a proline in the rhodopsin gene. The abnormal gene product kills the photoreceptor cells that express it. Previous experiments showed that ribozymes can discriminate and catalyze the in vitro destruction of mutant mRNAs derived from the transgenic RP rats.

"This is a clear example that you can make a ribozyme that doesn't do damage to a wild type gene in vivo and [that] knocks out the product of the mutant gene," Lewin said, "Basically what we are doing is using RNA to fight RNA."

The researchers used two different types of ribozymes, called "hairpin" and "hammerhead," which use different catalytic motifs to catalyze the same reaction.

To deliver the ribozymes, Hauswirth employed a modified a virus. Injections of this recombinant adeno-associated virus (AAV) vector carried the gene encoding the ribozyme into photoreceptor cells. This treatment slowed the progression of the disease for at least three months. Ribozymes lacking catalytic activity had less effect on retinal degeneration.

"These people have been exceptionally smart about choosing a target in which you have an autosomal dominant disease and in which inhibition of expression of the mutant form of the gene in any significant level has the potential to have a significant therapeutic effect for the patient," said John Burke, professor of microbiology and molecular genetics at the University of Vermont, in Burlington.

These results represent two firsts, according to Lewin: the first time a virus has been used to deliver a gene to treat an autosomal dominant inherited disease, and the first time ribozymes have been used to treat an inherited disease.

"If the results can be extended to humans with RP, this therapy would extend vision for years," Lewin wrote. The authors believe that a similar approach could be useful in the treatment of other autosomal dominant diseases such as Huntington's disease, myotonic dystrophy and Marfan's syndrome, provided animals models are available for testing.

"There really is no other approach [for treating] any other form of RP right now, except for maybe vitamin supplementation," Lewin said. The researchers plan to determine how long the beneficial effects of their gene therapy will last and whether it will benefit animals with advanced disease.

Scientists Seeking Biotech Partners

"Scaling from the mouse to humans is not trivial," Sieving warned. "What works in these animals may not work in humans."

That is why Lewin and Hauswirth intend to test their method on pigs with RP later this year. Pigs have eyes nearly the same size as humans'. Successful results in this animal model and demonstration of the safety of viral vector injections into primate eyes, could lead to Phase I clinical trials in the next few years. The authors are now contacting ophthalmologists with large caseloads in preparation for such clinical studies.

Lewin and Hauswirth are looking for biotech collaborators and have begun discussions with some companies. The University of Florida College of Medicine, in Gainesville, has applied for patent protection for part of the technology.

"I think there is an excellent possibility of treating humans with this approach," Sieving said. *