Development of cellular and animal models of coagulation factors deficiencies for the assessment of innovative therapeutic approaches acting on transcriptional and post-transcriptional regulation

In the last decades, enormous efforts have been pushed toward the development of molecular therapeutic approaches for human genetic diseases, and the research all over the world has obtained remarkable achievements, especially in gene therapy field. Notwithstanding, the intense research also led to potential therapeutic strategies based on the correction of the specific disease-causing defects, which might circumvent some limitations of gene replacement therapy. These approaches are of great interest for patients with coagulation deficiencies, since they would benefit even from small increase in functional protein levels. 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. The first part of this work deals with the usage of engineered transcription factors (eTFs) as potential therapeutic strategy for factor VII (F7) deficiency caused by two severe promoter mutations. Through the expression of gene reporter plasmids we created a cellular model for the 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 stimulating 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 levels in HepG2 cells and induced F7 expression in Hek293 cells that do not virtually express factor VII. The second part describes the exploitation of the Sleeping Beauty Transposon System (SBTS) to develop cellular and mouse model of haemophilia B (HB) caused by splicing mutations, in order to assess the efficacy of an RNA-based therapeutic approach. 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 minigenes assays. Therefore, the evaluation of the U1snRNA-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 preliminary studies provided us with optimized experimental protocol to create cellular models of human disease caused by splicing mutations. This also provides with the rationale for the creation of mouse models through hydrodynamic injection of the transposon plasmids and of the transposase in wt mice, for the assessment of the modified U1 snRNAs-mediated rescue in–vivo in a genomic expression context instead of a transient episomal system.