An Update on the Development of sd-rxRNA for Retinoblastoma Therapy

Abstract

Purpose: Retinoblastoma is a cancer that originates in the retina and primarily affects 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. 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 retinoblastoma therapeutic targets. Initial studies examined in vitro mRNA silencing in retinoblastoma cell lines as well as in vivo uptake by human retinoblastoma cells in a mouse model following treatment with control sd-rxRNA compounds. Here we designed and examined the efficacy of therapeutically relevant sd-rxRNAs specific for retinoblastoma in vitro.

Methods: Twenty-five sd-rxRNAs were designed, synthesized and screened in 3-point dose response studies in RB177 cells. Based on targeted mRNA reduction, potential hits were selected and further evaluated in 6-point dose response studies in RB177 and RB176 cells.

Results: Multiple sd-rxRNAs resulted in > 50% reduction of the targeted mRNA in studies carried out in RB177 cultured cells. Four potential hits were selected and further evaluated in 6-point dose response studies in RB177 and RB176 cells. Two candidate compounds that demonstrated > 50% mRNA target reduction at < 0.5 uM in both cell lines were selected (a lead and a backup) for in vitro evaluation of tumor cell viability post treatment and in vivo analysis in an orthotopic mouse xenograft model.

Conclusions: Therapeutically-relevant, target-specific sd-rxRNAs were identified and evaluated in vitro. Lead candidates were selected based on their ability to reduce the targeted mRNA in human retinoblastoma cell lines in vitro. Our next step is to evaluate if treatment with sd-rxRNAs results in reduced tumor cell viability in vitro and reduced target mRNA levels in human retinoblastoma cells in vivo in an orthotopic mouse xenograft model. 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.

Keywords: 703 retinoblastoma • 533 gene/expression