DEVELOPMENT OF AN IN VIVO MULTITASKING BIOLUMINESCENT REPORTER GENE PLATFORM FOR THE PRECLINICAL EVALUATION OF RNA THERAPEUTICS
RNA-based therapeutics have emerged as a transformative class of medicines, enabling precise modulation of gene expression through sequence-defined mechanisms and offering novel therapeutic opportunities for previously intractable diseases. The potential of these drugs to address unmet pharmacological needs, together with their novelty and pharmacokinetic and pharmacodynamic properties that often differ substantially from those of conventional small chemical molecules, makes their preclinical evaluation particularly challenging. Consequently, there is an urgent need for innovative preclinical platforms capable of capturing their complex biological effects beyond conventional toxicity and efficacy endpoints. This PhD thesis is aimed at developing and applying a reporter-based strategy to improve the rational design and preclinical evaluation of RNA-based therapeutics. By integrating in vitro reporter assays with in vivo bioluminescent reporter mouse models, this work enables non-invasive, longitudinal monitoring of key biological pathways involved in pato-physiological mechanisms including inflammation, oxidative stress, cell-cycle regulation, and immunoregulatory signalling. Firstly, in vitro reporter-based screening platform was established to assess off-target effects of the allele-specific siRNA siRyR2-U10, developed for the treatment of catecholaminergic polymorphic ventricular tachycardia (CPVT). Using multiple murine and human cell models, the effects of the siRNA and its calcium phosphate nanoparticle formulation were systematically evaluated across inflammatory, Th2 immune response, oxidative stress, and proliferative pathways. This multidimensional analysis revealed limited immunostimulatory and oxidative stress responses, mainly related to the calcium- phosphate nanoparticles formulation. In contrast, we identified a reproducible, sequence-dependent modulation of cell-cycle progression, thereby laying the groundwork for the use of high-throughput reporter assays based on general-purpose pathways as a preclinical approach to assess off-target effects. In the second part, a central innovation of this work is the development and application of a dual bioluminescent reporter system to resolve the spatiotemporal dynamics of early immune activation in vivo. The immunization process unfolds through a tightly orchestrated sequence of innate and adaptive events across distinct anatomical compartments; however, the early transcriptional signals that initiate these responses remain poorly characterized in vivo. To address this gap, complementary NFκB-luc2 and STAT6-luc2 reporter mouse models were implemented to enable real-time monitoring of pro-inflammatory pathway and Th2 immune response. A novel STAT6-luc2 reporter line was generated and validated to this purpose. When combined with the established NFκB-luc2 model, this dual-reporter platform enabled a comprehensive characterization of immune activation induced by anti-RidA mRNA vaccines both unmodified and N1-methyl-pseudouridine–modified sequences. This approach generated a spatiotemporally resolved atlas of vaccine-induced signalling, revealing formulation-dependent innate immune dynamics and identifying the liver as a dominant early hub for both NFκB and STAT6 activation following systemic administration. Integration of in vivo imaging data with downstream antibody responses demonstrated that transient early STAT6 activation correlates with robust humoral immunity, indicating that balanced and rapidly resolving innate signalling is a key determinant of effective vaccination. In contrast, pronounced abdominal NFκB activation was not predictive of improved vaccine performance. Together, these findings establish the dual NFκB/STAT6 reporter system as a rapid and predictive in vivo screening platform, capable of identifying promising vaccine formulations within the first 24 hours. More broadly, they uncover an underappreciated immunomodulatory role of the liver in mRNA vaccination and suggest that hepatic immune–metabolic cross-talk represents a critical regulator of downstream adaptive immune responses. Finally, a transcriptomic profiling of early mammary carcinogenesis was exploited to identify tumour-associated antigens expressed at premalignant stages and maintained during disease progression. Three candidates were selected as promising mRNA anticancer vaccines. In vitro evaluation demonstrated that these mRNAs effectively induce dendritic cell maturation, supporting their potential as anticancer vaccine candidates for ductal carcinoma in situ. Overall, this work demonstrates that reporter-based bioluminescent platforms provide powerful, integrative tools for the preclinical assessment of RNA-based therapeutics. By enabling dynamic, rapid and cost-effective in vitro and in vivo analysis, this approach is expected to support the development of next-generation RNA medicines.
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