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M. Moosajee, C. Y. Gregory-Evans, K. Gregory-Evans; Pharmacological Therapy Using Aminoglycosides Can Override the Genetic Defect Causing Choroideraemia in the Zebrafish Model. Invest. Ophthalmol. Vis. Sci. 2008;49(13):3069. doi: https://doi.org/.
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© ARVO (1962-2015); The Authors (2016-present)
Choroideraemia (incidence 1:50,000) is an untreatable X-linked recessive disorder characterised by a progressive chorioretinal dystrophy. Aminoglycosides, such as gentamicin, suppress disease-causing premature stop mutations, thus partially restoring the functional protein. A large proportion of choroideraemia patients harbour stop mutations in the REP1 gene, hence aminoglycoside therapy may provide a new treatment. The zebrafish model of choroideraemia (chmru848) used in this study has a recessive nonsense mutation in rep1 and displays ocular phenotypic features similar to human patients. The aim of this investigation is to identify an effective treatment for choroideraemia.
Mutant chm embryos were treated with 100 µM gentamicin or gentamicin-texas red conjugate, and 100 µM paromomycin from 8 hours post-fertilization; retinal histology and aminoglycoside entry into the retina was examined at day 6 using confocal microscopy. The chm mutation causes lethality by day 5; survival times were determined for the treated (n=50) and untreated (n=33) mutants. Rep1 protein was detected by Western blot analysis. Apoptosis in the retina was detected by TUNEL assay. Luciferase reporter assays for the chm mutation (Q32X) were performed to measure levels of readthrough with gentamicin and paromomycin.
Histological analysis of chm mutant embryos treated with gentamicin and paromomycin (LD50 of 300 µM and 280 µM respectively), showed significant rescue of retinal degeneration compared to untreated mutants at day 6, and that gentamicin effectively penetrated the retina. Treated chm embryos lived considerably longer (average 8.3 days) than untreated chm mutants (average 4.8 days); p<0.0001. Western blot analysis detected rep1 protein in treated chm samples. TUNEL assay revealed less apoptotic cell death in the retina of treated chm embryos at day 6. Luciferase reporter assays for both aminoglycosides induced readthrough of the premature stop codon.
This study provides ‘proof-of-principle’ for treating choroideraemia patients with aminoglycosides. Affected males tend to present during their teenage years complaining of night blindness with progression to complete loss of vision in middle age. Thus, early administration could arrest disease progression and even prevent this form of genetic blindness. It may also be applicable to other genetic eye diseases caused by nonsense mutations.
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