Robust Functionality and Regulation of Selectively Expressed RNA as AAV Vectors and In Vitro Transcribed Molecules

Background/Objectives: Selectively expressible RNA (seRNA) molecules represent a promising new platform for the induction of cell type-specific protein expression. Based on the sense-antisense interaction of the seRNA antisense domain with target cell-specific RNA molecules, the partial degradation of the seRNA molecule induces the activation of an internal ribosomal entry site to initiate translation. The selective expression of seRNA encoded proteins exclusively in target cells works both in vitro and in vivo but is associated with a lower expression intensity compared with classical mRNAs. Furthermore, seRNAs have so far been transfected into cells by plasmid-encoded seRNA expression systems, which is limiting their broad medical applicability. Here, we focus on the characterization of plasmid-based seRNA uptake and activation as well as on options to transfer the seRNA technology to additional vector systems to increase target cell-specific effector expression. Methods: seRNA constructs were generated as expression plasmids, AAV, DNA minicircles and IVT-RNA and delivered into different eukaryotic cell lines by transfection/transduction. Analyses were performed using fluorescence microscopy and, for quantitative analyses, flow cytometry. RNA stability and expression analyses were performed using qRT-PCR. Results: We show that seRNA-based plasmid systems are efficiently transfected into cells but that reduced RNA steady-state levels are present compared with control expression plasmids. This effect is most likely based on reduced transcription efficiency rather than seRNA stability. Furthermore, seRNA transcription from viral vectors or circular DNA significantly increased the effector expression of seRNAs and enabled linear expression regulation while maintaining target cell-specific activation and inactivation in non-target cells. Optimal results were achieved by adapting the technology to in vitro transcribed seRNA. Conclusions: Our data show that seRNA technology develops its full functionality regardless of the type of transfer vector used. Furthermore, expression strength can be regulated within a wide range while maintaining consistent functionality which will enable broad applicability in medicine in the future.