RNA activation (RNAa) is a mechanism in which small activating RNA (saRNA) interacts with the Argonaute 2 (AGO2) protein and the RITA complex to enhance the expression of target genes via binding to their promoter region. Due to their moderately enhancing effect on gene expression they hold great promise for application to therapy of rare, neurodevelopmental diseases where the affected genes are tightly regulated to function in a narrow physiological range with both insufficient expression and overexpression leading to a pathogenic phenotype. In this thesis we aimed to design single-stranded antisense oligonucleotides (ASO) based on the existing saRNA p21 and CEBPA in clinical trials in order to overcome delivery issues and immunogenicity associated with double-stranded saRNA. We then monitored their effect on target gene expression to see if they can mimick the effects of saRNA. With antisense oligonucleotides ASO p21 and ASO CEBPA we successfully activated expression of target genes as determined by mRNA quantification with qPCR and protein detection by western blot. As the saRNA activity depends on their interaction with AGO2 protein, we used immunoprecipitation and surface plasmon resonance (SPR) to monitor specific interaction of ASO and AGO2 which we also managed to confirm using SPR. Finally, based on the existing guidelines we designed new saRNA candidates targeting developmentally important genes ABCD1, ZC4H2, SYNGAP1 and CTNNB1, associated with rare, incurable, monogenic diseases and monitor their target gene activation by qPCR. With three of the five designed new saRNA we were able to show low target gene activation which will serve as the basis for further sequence optimisation and eventual ASO design.
|
