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N. L. Parmalee, K. Doi, J. Kong, J. Zernant, D. F. Schorderet, R. Allikmets; Controlled Suppression of RPE65 in vitro and in vivo: Implications for Treating Stargardt Disease. Invest. Ophthalmol. Vis. Sci. 2008;49(13):1698.
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© ARVO (1962-2015); The Authors (2016-present)
Stargardt disease results from accumulation of lipofuscin and its major fluorophore, A2E, in the retinal pigment epithelium (RPE). We tested the hypothesis that A2E accumulation can be reduced by slowing down the visual cycle by controlled suppression of the RPE65 gene via tetracycline-inducible RNA interference.
Short hairpin (sh)RNA sequences targeting the RPE65 gene were designed. Tet-inducible shRNA lentiviral vectors were made by cloning the silencing sequences into the lentiviral pLVCT-tTR-KRAB vector containing a GFP reporter gene and Tet-inducible H1 promoter. Viruses were produced and used to infect human ARPE-19 cells followed by induction of the shRNA with doxycycline. Knockdown of the RPE65 mRNA was confirmed by quantitative RT-PCR. Subretinal injections of concentrated virus were performed at postnatal day 5 in Abca4-/- mice. The shRNA expression was induced by supplying the animals with 2 mg/ml doxycycline in the drinking water starting at 2 months after injection. Treatment efficacy was monitored by SLO and ERG, and eyes were harvested for A2E quantification and histology.
Three shRNAs, targeting conserved sequences between mice and humans, were evaluated in the human ARPE-19 cell line and in the Abca4-/- mice. Integration of vectors in ARPE-19 cells was confirmed by visualization of GFP following induction with doxycycline. qRT-PCR results showed a 60%, 40% and 10% reduction in RPE65 mRNA transcripts for constructs 1-3, respectively. Abca4-/- mice were injected with the two more efficient viral vectors. Preliminary analysis at 3 months after induction with doxycycline showed a measurable reduction of ERG amplitude.
RPE65 expression was efficiently suppressed in the ARPE-19 cell line by two out of three shRNAs. No adverse effects were noted in treated Abca4-/- mice for up to 5 months and preliminary ERG results suggested an effect in suppressing the visual cycle. Ongoing analysis includes bi-weekly ERG measurements, quantification of A2E accumulation, and retinal histology.
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