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Michael Byrne, Hardeep Singh, Donglai Qi, James Cardia, Lakshmipathi Pandarinathan, Katherine Holton, Karen Bulock, Lyn Libertine, David Cobrinik, Pamela Pavco; Development of sd-rxRNA® for Retinoblastoma Therapy. Invest. Ophthalmol. Vis. Sci. 2013;54(15):1258.
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© ARVO (1962-2015); The Authors (2016-present)
Retinoblastoma is a cancer that originates in the retina and primarily affects young children. It is driven by RB mutations as well as the expression of genes involved in the “normal” signaling circuitry of retinal cells, particularly that of cone precursors. Some of these genes have been found to be critical to retinoblastoma cell growth and survival, suggesting that they may be effective therapeutic targets. We have developed a new class of stable, self-delivering RNAi compounds (sd-rxRNA®) that incorporate features of RNAi and antisense and results in spontaneous cellular uptake. Our goal is to use the sd-rxRNA platform to develop compounds against novel retinoblastoma therapeutic targets. Earlier studies of control sd-rxRNAs in the eye resulted in spontaneous cellular uptake in the retina as well as statistically significant reduction of target mRNA levels for up to 21 days. To determine the potential of using sd-rxRNAs to target genes involved in retinoblastoma, we examined in vitro mRNA silencing in retinoblastoma cell lines as well as in vivo uptake by human retinoblastoma cells in a mouse model.
For in vitro studies, retinoblastoma cell lines RB176, RB177 and Y79 were treated with sd-rxRNA targeting the PPIB gene for 48 hours, after which PPIB mRNA levels were analyzed by qPCR. For in vivo studies, 10 ug of DY547- labeled sd-rxRNA was administered by intravitreal injection to mouse eyes that were seeded subretinally 3 weeks earlier with Y79 cells.
In all retinoblastoma cell lines tested, PPIB was silenced in a dose-dependent manner at 48 hours post administration relative to a non-targeting control sd-rxRNA. Twenty-four hours post intravitreal injection in mouse eyes, sd-rxRNA was visible in tumor cells in the subretinal space and in the vitreous.
sd-rxRNAs reduce targeted mRNA levels in retinoblastoma cell lines. Additionally, sd-rxRNA is delivered to all cell layers of the retina and is taken up by tumor cells within 24 hours of intravitreal administration. These findings, along with our previous report of specific and extended silencing of retinal genes by sd-rxRNA, support the potential use of an sd-rxRNA treatment for retinoblastoma. Our next step is to design and characterize sd-rxRNA compounds against novel retinoblastoma targets and to evaluate their ability to reduce target mRNA levels in human retinoblastoma cells in vivo using an orthotopic mouse xenograft model.
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