Site-directed RNA Editing to Modulate Kinase Activity as a Chemotherapeutic

点击翻译按钮获取中文摘要

PROJECT SUMMARY The development of small molecules that target dysregulated protein kinases led to a paradigm shift in the treatment of cancer. However, the need for novel methods of kinase inhibition is demonstrated by selectivity challenges and the emergence of drug resistance. This proposal explores how RNA editing by Adenosine Deaminase Acting on RNA (ADAR) enzymes can be used to modulate target protein activity in cancers, through the optimization of guide oligonucleotides that promote editing in specific sequence contexts. The ADAR family of enzymes converts adenosine to inosine in RNA, and can be directed to a target adenosine by a complementary guide oligonucleotide. Thus far, the field of RNA editing by ADAR enzymes has focused on correcting disease-causative premature termination codons, due to ADAR’s preference for editing adenosines within a termination codon. However, lysine codons are known to be deaminated by ADARs, and are of particular interest due to the presence of a conserved catalytic lysine residue in protein kinases. Reaction of the lysine codon with ADAR2 would produce an arginine codon: a known inactivating mutation in protein kinases. To drive editing at the catalytic lysine within kinase mRNA, an established rational design approach to guide oligonucleotide chemical modifications will be used. Our strategy for site-directed RNA editing has high therapeutic potential because it utilizes endogenous ADAR enzymes, and therefore only requires the delivery of a guide oligonucleotide. While enzyme delivery faces barriers in efficiency and immune stimulation, oligonucleotide therapies have had recent successes in targeted delivery. The central hypothesis of this project is that guide oligonucleotide-directed editing of protein kinase mRNA will lead to a reduction in downstream cell signaling in cancers. Aim 1 will define how guide RNA modifications affect editing of target adenosines in lysine codons via oligonucleotide chemical synthesis, and in vitro activity assays. Aim 2 will evaluate the effect of protein kinase mRNA editing on protein phosphorylation and apoptosis in glioblastoma cells. Training in cell culture, Western blotting, and fluorescence microscopy will enable the cellular analysis of protein kinase mRNA editing. This proof of principle experiment will have broad implications in cancer therapeutics by establishing the efficacy of RNA editing to modulate disordered protein activity. More broadly this will expand the potential therapeutic applications of ADAR editing by defining how guide RNAs can be engineered to act in non-preferred sequence contexts.