Discovery may lead to Leber congenital amaurosis trials

Cleveland-Gene therapy and pharmacologic therapy, administered separately and combined, rescued retinal function in blind mice that were bred without an enzyme crucial to the detection of light.

These experiments are preliminary steps toward clinical trials in the treatment of a subset of cases of Leber congenital amaurosis (LCA) that could begin later this year or next year, said Krzysztof Palczewski, PhD, chairman of pharmacology, Case Western Reserve University, Cleveland.

The causes of LCA are not completely understood, but different forms of the disease may be due to abnormal development of photoreceptor cells in the retina, extremely premature degeneration of these cells, or lack of metabolic ingredients necessary for vision in the cells, according to Dr. Palczewski.

It is known that LCA can be caused by mutations in the gene encoding retinal pigment epithelium 65 (RPE65), a protein involved in production and recycling of a chromophore called 11-cis-retinal (11-cis-RAL) in the eye. About 15% of patients with LCA have a mutation in RPE65 that results in severely impaired rod and cone function. A study published several years ago showed that vision could be rescued in mice with RPE65 mutations.

The retina can also stop functioning due to the loss of the lecithin:retinol acyl transferase (LRAT) enzyme, which is necessary for the regeneration of a pigment necessary for the eye to detect light.

A third gene known to be involved in the production of chromophores and whose mutations can lead to LCA is retinol dehydrogenase 12 (RDH 12). Several other genes have also been identified that, if mutated, lead to LCA.

"We are at this very exciting time in biology that once such genetic findings are known, one can generate animal models that faithfully mimic human conditions, and test different pharmacologic approaches to rescue vision in these animals," Dr. Palczewski said. Results of the study of pharmacologic and gene therapy were published in the November 2005 issue of the open-access journal PloS Medicine.

In the study, investigators used knockout mice that were deficient in LRAT to test whether these therapeutic approaches could restore visual function. Besides testing each approach separately, they also studied the effects of combined therapy.

Oral treatment

In the oral treatment experiments, investigators determined through a series of tests of 9-cis-retinoids and esters that 9-cis-R-Ac and 9-cis-R-Su were most efficient in restoring visual pigment; 9-cis-R-Ac was used for the remainder of their tests. They subsequently observed that gavage with 9-cis-R-Ac produced a transient increase in retinoid levels in the liver and a more sustained increase in levels in the eye.

In a key finding, visual pigment was restored in the knockout mice 4 to 5 hours after gavage with 9-cis-R-Ac; these levels remained nearly constant for 96 hours. Electroretinographic (ERG) and pupillary light responses (PLR) also were significantly restored.

"At 8 o'clock in the morning you have blind mice. When you take this compound (9-cis-R-Ac) dissolved in vegetable oil and give it to mice orally, before lunch they recover vision almost as good as in wild-type mice," Dr. Palczewski said.

"The reason is because mammals develop a very elaborate and efficient way of extracting vitamin A from food and delivering it throughout the body, particularly to the eye," he explained. "We are hijacking that delivery system from the stomach to the eye. We deliver the precursors to the eye in a few hours, and then they become further metabolized and recombine with rhodopsin and cone opsins. The excess of this drug is washed out because LRAT is also expressed in other tissues but lack of LRAT prevents accumulation of retinoids."

ERG response increased from about 5% of wild-type levels in the LRAT mice to about 50% of wild-type levels in treated mice (p < 0.05 versus wild-type and knockout controls). Visual pigment increased from undetectable levels to 600 pmoles per eye in mice treated with the retinoid. Improvement of about 1,000-fold was seen in PLR and ERG responses.