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Future Prospects and Hopes in RPE65 Gene Therapy
In the late 80's (10), (9), (3) a protein that is found only in the RPE and whose apparent molecular weight is approximately 65 kDa was discovered and named RPE65. RPE65 is involved in the isomerization step of the visual cycle as indicated by the absence of the 11-cis retinol in mice lacking RPE65 (20) but its precise function remains unknown. Later on, its human gene was cloned (17) and mutations discovered in cases of severe retinal dystrophies starting in early childhood, often diagnosed as Leber's congenital amaurosis (LCA) , specifically called type 2 (14), (16) or severe childhood-onset retinal dystrophy (7). There are also a few less severe cases (15), (16) whose time course is similar to that of retinitis pigmentosa (RP). Although affected children are severely handicapped, they are not blind (18), (12), (19), (21), (8) suggesting that alternative pathways may minimally compensate for the RPE65 deficit. Unfortunately, the disease is rapidly evolving and the progressive loss of rods and cones leads usually to blindness at the adult stage. Screening of large patient samples (16), (13), (21) indicates mutations of RPE65 in 7 to 10 % of LCA cases and 1 to 2 % of recessive RP cases. This would account for roughly 2 to 4 RPE65 patients per million individuals in developed countries.
In 1998, it was discovered that the congenital night blindness of the Briard shepherd , a large dog originating from Brie in France, is due to a mutation in Rpe65 (2), (23) . Previous studies (26), (25) showed that dog puppies undergo a severe rod and cone , and to a lesser extent cone dysfunction from birth on, but that the photoreceptors disappear very slowly during the dog life. Nevertheless, the dogs are totally night blind and also show impairment in day vision. When human mutations were found in 1997, several therapeutic strategies were considered. One way of circumventing the RPE65 deficit was to provide 11-cis retinal to the photoreceptors. A group of researchers from the University of Washington in Seattle fed Rpe65 -deficient mice with 9-cis retinal (that can replace 11-cis retinal) and showed that rod visual pigment and function were rapidly restored (22) . These results confirmed that the RPE65 deficit is linked to the lack of cis-retinoids and suggested that in young animals the degenerative process could be reverted. However, long term efficacy of such a treatment and toxic effects due to the accumulation of retinol in the retina are unknown. Another way to cure for the RPE65 deficit was to perform RPE transplantation from normal to Rpe65 -deficient mice. This strategy has now being tested for 2 years (6) and results are still awaited. Although an attractive alternative to cis-retinoid feeding, it will require delicate surgery to allow for proper engrafting of RPE cells on a relatively large surface. In addition, application to humans will need to resolve the problem of graft rejection which is likely to occur since RPE cells are immunogenic.
A third way of treatment was gene therapy, that is to introduce a normal copy of the RPE65 gene into RPE cells, integrate it in the host genome, and allow for the life time production of the normal RPE65 protein. This is what has been tried by a group of researchers from USA (1) . The RPE65 gene was cloned in rAAV , a non pathogenic virus that greatly facilitates the penetration of the gene in RPE cells and its integration to the genome of the cells. Three 4-month old Briard dogs received an injection of the AAV-RPE65 in the eyes, in some eyes in close contact with RPE cells (subretinal injections) and in other eyes at a distance from RPE cells (intravitreal injection). Subsequent analysis showed a significant, light induced-retinal activity in eyes injected subretinally while uninjected or intravitreally injected eyes had no responses to light. Moreover, the dogs with subretinally injected eyes displayed avoidance behaviour to obstacles in dim red light indicating that they were seeing the environment while an untreated dog of the same age was blind in this light conditions.
Previous experiments have shown successful gene therapy in rodent models of retinal degeneration. This study demonstrates that retinal gene therapy is also efficient in a large model, the Briard dog, whose eyes are about the size of human's. This is extremely encouraging since it suggests that gene therapy can be a treatment for human retinal degenerative diseases. In the case of
RPE65
, this would apply to
Leber's congenital amaurosis type 2
and some forms of
early onset retinitis pigmentosa .
However, before performing human trials, one should realize that additional experiments must be done. The treated dogs will be kept for several years in order to examine whether or not the beneficial effect is long lasting. Other dogs will be treated; some of them receiving various doses of
AAV-
RPE65
, others having injections in several parts of the retina in order to cover the whole surface of the retina. In addition, mice will receive injections in the eye or elsewhere in the body and they will be checked for toxic effects or possible invasion in the germ line. If these experiments show that the treatment is safe and when the optimal doses of virus have been determined, patients will be recruited for trials.
Only some patients could participate. Indeed, in order for the treatment to be efficient, a reasonable number of photoreceptors must still be present in the retina, implying that the disease should not have evolved too much. The point of no return beyond which this type of gene therapy is not efficient will have to be determined. In the particular case of retinal degeneration due to
RPE65
mutations, the point of no return may be relatively late because we know that photoreceptors, even though they are not functioning, remain present in the retina several years after birth. In other retinal degenerations due to mutations in rod photoreceptor-specific genes with early loss of rods, this type of gene therapy may be restricted to very young people.
Gene therapy, in the case of loss-of-function of a gene defect, the situation most frequently encountered in recessive diseases like RPE65 retinal degeneration, is based on transferring one normal copy of a gene in the defective cell. However, gene therapy may be conceived in other ways. One can transfer a gene whose product (ribozyme) will specifically recognize and hydrolyse the mutated transcript from one of both alleles of a gene that is harmful for the cell (a situation found in some dominant diseases) while sparing the other normal transcript of the other allele that will be enough for the function. Gene therapy may also be useful to produce neuroprotective agents in some retinal cells, in order to prevent or slow down the degeneration of photoreceptors. Such a strategy may apply to patients at a later stage of degeneration. The RPE65 story is probably at the beginning of a long chain of gene therapy trials in the field of retinal degeneration that promises great hopes for patients and their families.
References
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Human Mutation Online
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Dr. rer. medic. Markus Preising, Dipl.Biol. Molecular Genetics Laboratory Department of Paediatric Ophthalmology, Strabismology and Ophthalmogenetics University of Regensburg Head: Prof. Dr. med. Birgit Lorenz |
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