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• W960773756 abstract "Figure 5. MDM2-targeting sd-rxRNAs Significantly Reduce MDM2 mRNA Levels in vitro Through Day 6 Conclusions • MDM2 is known to be a crucial component of the cone circuitry underlying retinoblastoma development and is critical to sustained expression of the MYCN oncogene. • MDM2-targeting sd-rxRNAs were identified that reduce MDM2 mRNA and protein levels in vitro. Importantly, MDM2targeting sd-rxRNA treatment of RB176 cells resulted in reduced MYCN protein levels over time. • Treatment of RB176 cells with MDM2-targeting sd-rxRNAs resulted in reduced tumor cell viability over time. • These findings support the potential use of MDM2-targeting sd-rxRNA for retinoblastoma therapy. Background Figure 1: Mechanism of Cellular Gene Silencing RNA interference (RNAi) is a naturally occurring cellular process. Introduction of double stranded RNA into cells can result in association with the RNA-induced silencing complex (RISC) to target complementary mRNA sequences and degrade target genes. Figure 2: sd-rxRNA Incorporates Advanced Features of RNAi and Antisense Technologies Figure 4: MDM2-Targeting sd-rxRNA Selection E-mail mbyrne@rxipharma.com for PDF version of the poster sd-rxRNAs are asymmetric RNAi compounds comprised of a small duplex region (<15 base pairs) and a single-stranded phosphorothioate tail (≥ 6 nucleotides). In addition, sd-rxRNA compounds are chemically modified with stabilizing and hydrophobic modifications (e.g., sterol), which confer stability, efficient cellular uptake and reduced inflammatory response. RB177 cells (50,000 per well) were treated with 0.05, 0.1 or 0.5 uM of compound. Seven compounds with ~40-50% reduction of MDM2 mRNA at 0.5 uM were identified. Four compounds were chosen for 6-point dose response studies in RB176 and RB177 cell lines. At 48 hours post administration MDM2 mRNA levels were quantified by a branched DNA assay. From these screens two compounds (25799 and 25805) were chosen for further analysis. NTC= non-targeting sd-rxRNA. Figure 3: sd-rxRNA: Dose Dependent Silencing in vitro in Retinoblastoma Cell Lines Single compound incorporates activity and delivery Robust uptake & silencing in multiple preclinical models Structural diversity = novel intellectual property Combining many positives of RNAi & antisense, while avoiding many negatives Provides for broad pipeline of RNAi drugs for unmet medical needs Purpose: Retinoblastoma is a cancer that originates in the retina of young children. It is driven by inactivating RB1 mutations, as well as by 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. RXi has 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 retinoblastoma therapeutic targets. We selected MDM2 as a target because it is a crucial component of the cone circuitry underlying retinoblastoma development and is required to sustain expression of the MYCN oncogene in retinoblastoma cells. Initial studies demonstrated in vivo uptake of control sd-rxRNA by human retinoblastoma cells in a mouse model and identified candidate sd-rxRNAs that down-regulated MDM2 in cultured retinoblastoma cells. Here, we evaluated two MDM2-targeting sd-rxRNAs for effects on MDM2 mRNA and protein expression, as well as impact on MYCN protein expression and cell proliferation. Methods: Two MDM2-targeting sd-rxRNAs identified from screening studies were evaluated in cultured RB176 cells. MDM2 mRNA was evaluated using a branched DNA assay. MDM2 and MYCN protein levels were determined by Western blot. Cell proliferation was evaluated by assaying for ATP production. Results: MDM2-targeting sd-rxRNAs reduced MDM2 mRNA levels as well as MDM2 protein levels over time. Importantly, treatment with MDM2 targeting sd-rxRNAs also decreased MYCN protein expression and cell proliferation, suggesting a potential therapeutic impact on retinoblastoma cell viability. MDM2 mRNA levels were significantly reduced in a dose dependent manner through Day 6. Moreover, MDM2 and MYCN protein levels were reduced through Day 7. Conclusions: MDM2-targeting sd-rxRNAs were evaluated in vitro. Target-specific mRNA and protein reductions were observed. Treatment of cultured cells with MDM2 targeting sd-rxRNAs also led to reduction of MYCN protein levels which importantly can lead to retinoblastoma cell death. These findings, along with our previous report of specific and extended silencing of retinal genes by sd-rxRNA, support the potential use of sd-rxRNA for retinoblastoma therapy. 0 0.2 0.4 0.6 0.8 1 1.2 PPIB sd-rxRNA NTC 1 uM 0.3 uM 0.1 uM 0.05 uM 0.025 uM 0.01 uM 0 0.2 0.4 0.6 0.8 1 1.2 PPIB sd-rxRNA NTC 0 0.2 0.4 0.6 0.8 1 1.2 PPIB sd-rxRNA NTC PP IB m RN A Le ve ls R el at iv e to N TC (N or m al ize d to G AP DH ) RB176 Y79 RB177 Model sd-rxRNAs were designed to target PPIB, a ubiquitously expressed gene. 50,000 cells per well were treated with PPIB targeting sd-rxRNAs in vitro at 0.01, 0.025, 0.05, 0.1, 0.3, and 1 uM. At 48 hours post administration PPIB mRNA levels were reduced in a dose dependent manner relative to non-targeting control (NTC) sd-rxRNA. mRNA levels were quantified by a branched DNA assay. sd-rxRNA sd-rxRNA/Cone arrestin Cone arrestin a c b c a b Figure 3: Uptake of sd-rxRNA in vivo in Mouse Retina and Tumor Cells 24 hours Post Intravitreal Injection Mouse eyes were seeded subretinally with Y79 cells. Three weeks post seeding, 10 ug of fluorescent sd-rxRNA (red) was administered by intravitreal injection. Twenty-four hours post injection a) sd-rxRNA (red) co-localized with tumor cells (green) in the subretinal space; b) sd-rxRNA localized with tumor cells in the vitreous; c) sd-rxRNA is visible in the retina. 0 20 40 60 80 100 120 14" @default.
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• W960773756 date "2015-06-11" @default.
• W960773756 modified "2023-09-24" @default.
• W960773756 title "MDM2 Targeting sd-rxRNA® for Retinoblastoma Therapy" @default.
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