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P. H. Tang, J. Fan, P. W. Goletz, L. Wheless, R. K. Crouch; Matrigel as a Novel Vehicle for Sustained Delivery Of 9-cis Retinal to Rescue Cone Photoreceptors in Rpe65-/- Mouse Pups. Invest. Ophthalmol. Vis. Sci. 2009;50(13):1215.
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© ARVO (1962-2015); The Authors (2016-present)
Leber’s congenital amaurosis (LCA) is caused by mutations in the gene coding for RPE65 protein, essential for generating 11-cis retinal for the visual cycle. Absence of a normal cycle leads to early onset photoreceptor degeneration. Gene therapy has recently been shown to have potential for treating congenital eye diseases; however the difficulties of administering it to children diminishes its value as a stand-alone method for treating LCA. Developing alternative therapies to preserving cones in young LCA patients is crucial until they grow to tolerate gene therapy. The purpose of this study is to utilize Matrigel as a model for sustained 9-cis retinal delivery to RPE65-/- mouse pups for early cone rescue.
RPE65-/- mouse pups were treated with 9-cis retinal by mixing the retinoid with Matrigel and injecting subcutaneously at ten days after birth (P10). The efficacy of Matrigel as a delivery vehicle was compared against IP injections at P10, P14, P18, P22 and P26 as well as Matrigel-vehicle control. Animals were placed in constant dark after initial injection and analyzed at P30 using ERG response to test photoreceptor function. FITC-PNA-Lectin stained retinal flatmounts were analyzed for cone counts, and immunostaining of cross sections were used to determine protein localization.. For quantification of retinoids using HPLC, 2 month old mice were subcutaneously injected with 9-cis retinal in Matrigel and compared against IP-injected and Matrigel vehicle control mice and underwent a bleaching protocol seven days after injection (T7). At T15 animals were sacrificed and eyes were processed for retinoid quantification.
ERG analysis showed an increased cone response in Matrigel-treated animals as compared to IP-injected and control animals. Quantification of cones present in FITC-PNA-lectin stained retinal flatmounts showed 50% more cones in Matrigel-treated animals than IP-injections, a finding confirmed through immunohistochemistry cross sections. Retinoid quantification with HPLC showed 40% increase in 9-cis-retinal levels in the retina of Matrigel-treated as compared to IP-injected and vehicle control animals.
These results suggest that our Matrigel-based model for sustained delivery of 9-cis retinal is an effective method to preserve cones in the young RPE65-/- mouse pup. This provides a novel tool for research as well as a possible therapeutic development.
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