Development of cellular and animal models of coagulation factor deficiencies for the assessment of innovative therapeutic approaches

Autore del lavoro candidato: Elena Barbon

SINTESI CONTENENTE UNA BREVE DESCRIZIONE DEL LAVORO SVOLTO E DEI RISULTATI OTTENUTI: This work propose the development of in-vitro and in–vivo models of rare bleeding disorders, in order to explore corrective molecular approaches acting on the specific disease-causing defects, both at transcriptional and post-transcriptional level. Part of this work deals with the usage of engineered transcription factors (eTFs) as potential therapeutic strategy for factor VII (F7) deficiency caused by severe promoter mutations. Through the expression of gene reporter plasmids we created a cellular model for two F7 promoter variants. Then, we assembled four eTFs (TF1-4) designed to target different regions on the F7 proximal promoter in order to test their efficacy in stim ulating transcriptional activity on the target gene. The treatment with the different eTFs demonstrated that TF4, targeting a sequence between the mutations, induced a robust increase of gene transcription in the presence of the defective promoter. Interestingly, TF4 appreciably increased the endogenous F7 transcription and mRNA and protein levels in hepatoma HepG2 cells. The second part of the research activity was focused on the exploitation of the Sleeping Beauty Transposon System (SBTS) to develop cellular and mouse model of coagulation factor IX deficiency, haemophilia B (HB). In particular, we aimed to create models of HB caused by splicing mutations, in order to subsequently assess the efficacy of an RNA-based therapeutic approach. In fact, in the last years modified small nuclear RNAs U1 (U1snRNAs) have been exploited to correct splicing mutations causing severe coagulation factor VII deficiency and HB, but only in minigene assays. Therefore, the evaluation of the U1 snRNA-mediated correction strategy in–vivo implies the creation of proper mouse models for each specific splicing-variant, not yet available. Here we used the SBTS to develop cellular/mouse models of HB caused by the factor IX ex5-2C splicing variant. We have generated HEK293 stable clones expressing the normal or mutated human splicing-competent factor IX cassettes integrated into the genome as a result of the transposase activity. These studies provided us with optimized experimental protocol to create cellular models of human disease caused by splicing mutations. This also provided with the rationale for the creation of mouse models through hydrodynamic injection of the transposon plasmids and of the transposase in wild type mice, for the assessment of the modified U1 snRNAs-mediated rescue in–vivo in a genomic expression context instead of a transient episomal system.