Development of novel oligonucleotide therapeutic molecules targeting microRNAs for tackling brain cancers

Leon Maria Larcher

MicroRNAs are short non-coding RNA molecules ( ̴ 22 nucleotides) which are important in regulating numerous metabolic and cellular pathways responsible for cell proliferation, differentiation and survival. Due to the malignant nature of many cancer types, conventional treatment approaches face a number of challenges. In Glioblastoma Multiforme (GBM) localisation in the brain results in additional treatment challenges, such as, limited capacity for the brain to repair itself after treatment, ineffective drug delivery through the blood brain barrier, and neurotoxicity of chemotherapy. MicroRNA signature have been shown to be dysregulated in cancers. Interestingly, restoration of these dysregulated miRNA have been shown to abrogate and even reverse malignant phenotypes. In addition, it has been shown that miRNAs contribute to responses to drug therapy and are themselves modified by these drug therapies. There are a number of approaches targeting miRNAs that have shown great promise in cancer therapy. However, there are still key challenges that need to be overcome before the wide spread use of these technologies. In this project we develop the use of synthetic catalytic oligonucleotides (DNAzyme), which can bind and cleave target miRNAs. Following cleavage, the DNAzyme is free to continue their action, thus reducing the quantity of molecule needed to have a therapeutic effect. Two miRNA targets were selected in our study, namely, miR-21 and miR-494, which were selected following a comprehensive review of known miRNAs in GBM from the literature, miR-21 and miR-494 were selected due to their high overexpression and oncogenic roles (roles in growth, proliferation, invasion, and apoptosis) within GBM. In vitro cleavage analysis, revealed effective cleavage of the miRNA precursor (pre-miRNA) strands by three DNAzymes in a dose-dependent manner. In cell (U87MG, MDA-MB231) cleavage analysis revealed efficient knockdown of miR-21 using RNV541, however an increase in expression was observed when targeting miR-494 in cell (Huh-7) using RNV537 and RNV538. Concentration, structure, length, cell type and transfection reagent used can all influence transfection outcomes. Overall, the project showed that DNAzymes RNV541 efficiently cleaved miR-21 in vitro and inhibited the expression in cell. However cleavage of miR-494 was inducible only in vitro using both RNV537 and RNV538.

Development of novel oligonucleotide therapeutic molecules targeting microRNAs for tackling brain cancers

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