RNA-BASED THERAPEUTIC APPROACHES FOR BLOOD COAGULATION FACTOR DEFICIENCIES CAUSED BY SPLICING MUTATIONS

BACKGROUND - Gene mutations that generate Defective Donor Splice Sites (DDSS) and induce aberrant pre-mRNA splicing represent a frequent cause of human genetic disease. By using coagulation factor VII (FVII) gene as pioneer model, we previously demonstrated in cellular models that targeting DDSS by modified U1 small nuclear RNAs (U1snRNAs) significantly rescues FVII function gene impaired the IVS7+5g/a mutation. In this project we explored U1snRNAs as potential therapeutic tool for coagulation factor deficiencies such as FVII and factor IX (FIX, Hemophilia B) deficiencies that may lead to life-threatening bleeding and have still significant unmet medical needs. RESULTS - By exploiting cellular models we characterized the pathogenic molecular mechanism of several mutations producing DDSS in FVII and FIX genes. Appropriately engineered U1snRNA were able to rescue pre-mRNA splicing and the synthesis of procoagulant proteins to an extent that, if translated in patients, would be therapeutic. Moreover, we developed a novel correction strategy based on Exon Specific U1snRNA (ExSpeU1s) targeting non-conserved intronic sequences downstream of the donor site and demonstrated that a unique ExSpeU1 can rescue exon skipping caused by different mutations at the DDSS but also in the acceptor splice site and in the exons. For the first time, by taking FVII deficiency caused by the IVS7+5g/a mutation as model, we demonstrated that the U1snRNA-mediated approach results in appreciable and sustained levels of circulating FVII in mouse models. Finally, ExSpeu1 have been proved to correct splicing mutations associated to Cystic Fibrosis and Spinal Muscular Atrophy. PERSPECTIVES - These data encourage further studies with ExSpeU1 in animal models aimed at assessing efficacy and safety. The U1snRNA mediated approach might represent a valuable therapeutic tools for human genetic disease, and particularly for those in which replacement gene therapy is hardly approachable.