The aim of this proposal is to clarify the molecular mechanisms of a gene targeting approach called Small Fragment Homologous Replacement (SFHR). This approach can stably modify a genomic sequence by homologous replacement of a small DNA fragment and could correct mutations within disease genes. SFHR mechanism is poorly understood and its practical application is limited by a low and variable frequency of correction.
We already published experimental evidences 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. Both the general pathways and the related specific genes are excellent targets for SFHR molecular mechanism study and manipulation.
This proposal will focus on the relationship between the 4 pathways listed above and SFHR, with particular regard to the enhancing of correction efficiency. This relationship will be studied in human Cystic Fibrosis (CF) cells of airway epithelium. Both primary differentiated and long-term stem-induced (by the culture reprogramming condition - CRC - approach) CF airway epithelial cells will be used. Drugs acting on each of the 4 pathways and single gene targeting will be used to dissect the pathways and to manipulate SFHR efficiency. A possible enhancement of SFHR by the use of CRISPR/Cas9 method will also be evaluated.
These studies will contribute to a better comprehension of the molecular mechanisms of homologous genomic replacement after the uptake and intracellular processing of exogenous DNA. The selection of molecular targets to manipulate will provide suggestions for increasing gene repair efficiency. The application to both differentiated and stem-induced cells may highlight new perspective for SFHR therapeutic applications, in particular to CF.
The most innovative aspects of this proposal are the following.
1) The application of SFHR and CRISPR/Cas9, both innovative approaches of gene targeting, and their synergistic use.
2) The application of these approaches to a complementary combination of innovative cellular models; in particular to CF long-term stem-induced airway epithelial cells (CF-CRC-AESC) suitable for ex vivo correction and cellular therapy.
3) The fact that the evaluation of the interconnection between gene targeting (both SFHR and CRISPR/Cas9) and DNA methylation, chromatin remodelling, DNA repair and cell cycle pathways are an almost unexplored field.
4) The use of selected genes to be manipulated for the enhancement of gene targeting.
The results of our studies will contribute to the comprehension of the molecular mechanisms of recognition and cellular processing of exogenous DNA. They will allow an advancement of knowledge about gene targeting, focused on the enhancement of its correction ability. This will constitute a further step towards the possibility of correction of a mutation directly within the genome of patients. In particular about CF, the spin-offs are both the possibility of direct correction in the lung of patients, as well as the ex vivo approach as cellular therapy. In this case, the aim is to pick-up cells from the patient, reprogram them to stem cells (possibly easier to correct), correct and re-administer them to the patient after correction, to complement the action of diseased cells. Crucial effectors and gene-specific targets to be manipulated for the enhancement of SFHR efficiency, in particular in CF, will be revealed. This can allow the achievement of a higher and more specific correction for a practical SFHR and/or CRISPR/Cas9 method application to both in vivo and ex vivo therapeutic approaches.