REPorter system for RNA-based therapy detecting cellular stress in ORGanoid models - REP-ORG systems

Introduction: The progress in understanding the regulatory mechanisms of RNA has led to the development of innovative therapies capable of modulating gene expression underlying various diseases. Despite their great potential, these strategies still face limitations due to the multifunctionality and structural malleability of RNA molecules. Therefore, new methods are needed to improve both delivery accuracy and therapeutic effectiveness. This study is part of a research project aimed at standardizing a system for analyzing the pharmacodynamics of RNA-based drugs, with a particular focus on identifying off-target effects. In this context, we have generated reporter organoid models to assess the long-term effects on non-specific targets. Methods and Results: We generated constructs containing fluorescent proteins, GFP and YFP, under the control of consensus sequences recognized by transcription factors involved in the inflammatory response (NF-kB) and oxidative stress (Nrf2). These constructs were stabilized in cell lines derived from different tissues, including muscle (C2C12 and C2SOD11 lines), liver (HepG2 and HUH7), thyroid (Nthy-ori, K1, and BCPAP), and pancreas (MIA PaCa-2 and PANC-1). The proper functioning of the constructs was validated in 2D cultures treated with hydrogen peroxide by analyzing the activation of NF-kB and Nrf2 through Real-Time PCR and Western blot. Subsequently, the transfected cells were cultured using extracellular matrix. The organoids obtained were chronically treated with anti-RYR2 siRNA2, the molecule under study in the PNRR project, which targets the mutant allele of the RYR2 gene, the cause of catecholaminergic polymorphic ventricular tachycardia (CPVT). The organoids were monitored using a fluorescence microscope, and the fluorescence was quantified with the Varioskan. When fluorescence appeared, indicating activation of cellular stress pathways, we collected the organoids and analyzed the expression of two genes, RUFY3 and RPGR, potential off-targets identified through in silico analysis of the siRYR2-U10 sequence, using Real-Time PCR. Conclusions: We have created the REPorg systems to standardize the analysis of adverse effects caused by RNA-based drugs. This method allows for the detection of aberrant gene expression and off-target effects by utilizing fluorescence activation triggered by cellular stress. Additionally, these systems help determine the maximum tolerated dose of RNA-based drugs in cells.