Evaluation of the biodistribution and activity of new optimized readthrough molecules in Cystic fibrosis mouse model and other model systems

Nonsense mutations cause approximately 11% of inherited diseases, including Cystic fibrosis (CF), Duchenne Muscular Dystrophy (DMD), and some Primary Immune Regulatory Disorders (PIRD) can be mentioned. Nonsense mutations, also known as stop mutations, result in the presence of a premature termination codon (PTC) in the mRNA sequence, leading to premature translation termination. The consequence is the production of a truncated and non-functional protein, which is degraded. Simultaneously, the nonsense-mediated pathway (NMD) is activated, eliminating the available mRNA pool for translation. Currently, there is no cure for this genetic defect, resulting in life-threatening pathologies, such as CF or PIRD. CF is specifically caused by mutations in the gene encoding the Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) protein. About 2500 different mutations are associated with CF with the most common being a three-base pair deletion causing the loss of phenylalanine at position 508 (ΔF508). Nonsense mutations in the CFTR gene represent approximately 10% of CF cases, resulting in the absence of functional protein and a more severe form of the disease. Apart from symptomatic approaches to care for CF patients, a pharmaceutical strategy targeting the specific genetic defect has been pursued. Heterocyclic scaffolds play a crucial role in the personalized medicinal approach, as demonstrated by numerous studies in the CF field. Similarly, PIRD is a heterogeneous group of immune diseases caused by 430 genes, among which Lipopolysaccharides (LPS)-Responsive Beige-like Anchor (LRBA) has been identified. The LRBA deficiency disease has been recently described, highlighting its multiple effects due to the ubiquitous localization of the LRBA protein, e.g., immune cells and endocytosis vesicles. Concerning nonsense mutations, in the last years, the experimental approach proposed for these genetic alterations is the premature termination codon (PTC) translational readthrough (TR) by small molecules. These molecules promote the bypass of the PTC, allowing the production of a full-length protein. The current proposal aims to study three new Translational Readthrough-Inducing Drugs (TRIDs) (NV848, NV914, and NV930) in a model system for CF and LRBA-PIRD. The first part of the project was focused on CF and involved both in vivo and in vitro systems. After evaluating the acute toxicity in vivo of the three NV molecules, attention was focused toward NV848, to study the biodistribution in a wild-type mouse model and the efficacy in a CF-nonsense murine model. Additionally, CF-nonsense-engineered cell lines (16HBEge) were used to assess if CFTR functionality was restored under NV848 treatment, utilizing the Ussing chamber technique. The project's second part aimed at rescuing the LRBA protein in a specific nonsense mutation using the three NV TRIDs. The study specifically investigated the TRIDs’ effectiveness on LRBA expression, and functionality in human primary fibroblasts carrying the stop mutation c.5047C>T (p. Arg1683*) after prolonged treatments with the TRIDs.