In this project the researchers sought to use an alternative or adjuvant approach to the use of existing modulators, potentiators and correctors, or combinations thereof. The rationale of the project was to increase the amount of CFTR protein, even if mutated, with the aim of compensating for the decreased functionality with the increase in amount. To do this we decided to act on two aspects of gene expression: 1) on the gene promoter, DNA sequence recognized by protein complexes that control how many copies of the CFTR gene must be made (transcribed into mRNA); 2) on specific sequences of CFTR mRNA targeted by small RNAs (microRNAs) which usually cause a decrease in protein production.
On point 1, we built a plasmid construct capable, once delivered inside the cells, of producing a complex of proteins and RNA molecules to recognize the CFTR promoter, activating its transcription and increasing the mRNA copies. This complex consists of a modified CAS9 protein, unable to cut DNA but fused to a trans-activator factor. This protein is expressed in cells together with a guide RNA to recognize the CFTR promoter, specifically activating and/or increasing its transcription and therefore the amount of final CFTR protein.
As regards point 2, we used artificial molecules (PNA – Peptide Nucleic Acid), similar to DNA molecules, but modified by replacing the sugar-phosphate backbone with amino acids, which make them particularly stable. The purpose of these molecules was to counteract the action of the microRNAs, therefore, to stabilize the CFTR mRNA, avoiding its degradation. Therefore, although from different points of view, these two approaches have made it possible to increase the amount of expression of the CFTR gene, paving the way for the development of molecules capable of increasing the amount of the CFTR protein, which although mutated, would serve as a substrate for the current modulators to improve their clinical effect.
A further important result was to develop a delivery system for these molecules, capable of working also for other types of molecules, based on the use of PLGA (poly lactic glycolic acid) nanoparticles, vectors capable of transporting small molecules even in aerosol form and already in clinical use.