Ricerc@Sapienza

The aim of this proposal is to clarify the molecular mechanisms of the Small Fragment Homologous Replacement (SFHR) gene targeting approach. SFHR can stably modify a genomic sequence by homologous replacement of a small DNA fragment. This approach can potentially correct mutations within disease genes. The practical application of SFHR is limited by a low and variable frequency of correction, mainly depending on the poor knowledge of underlying mechanisms.
We already published the results about the interconnection between SFHR, DNA methylation, chromatin remodelling, DNA repair and cell cycle pathways. We also published a selection of 18 specific genes within DNA repair and cell cycle pathways involved in the SFHR. These general pathways and the related specific genes appears to be excellent targets for SFHR molecular mechanism study and manipulation, aimed to the enhancement of correction efficiency.
This proposal is the continuation of the previous funded project and will focus on the relationship between the 4 pathways listed above and SFHR. These studies will be performed in a reporter cellular system of mouse embryonic fibroblasts and in human Cystic Fibrosis (CF) cells of airway epithelium. Long-term stem-induced CF airway epithelial cells, obtained by the culture reprogramming condition approach, will be used. Also the differentiated counterparts of stem-induced CF cells will be used. Drugs acting on each of the 4 pathways, single-gene targeting and the CRISPR/Cas 9 method will be used to dissect the pathways, clarify SFHR mechanisms and enhance the correction efficiency.
These studies will allow a better comprehension of the molecular mechanisms of homologous genomic replacement. The selected molecular targets will provide suggestions for increasing gene repair efficiency. New perspectives for SFHR therapeutic applications to both differentiated and stem-induced cells will arise, in particular for the mutation-specific treatment of CF at DNA level.