Genome editing is a novel technology that allows repairing genetic mutations, including alterations in the CFTR gene which cause cystic fibrosis. Nonetheless, current technology does not allow repairing all types of mutation, such as the F508del which comprises three nucleotides deletion. Aiming at overcoming the limitation imposed by current technology, we turned to point mutations (single nucleotide) namely “neutralizing” mutations, that have been reported generating a milder F508del phenotype. The researchers initially tested the efficacy of the “neutralizing” mutations by analyzing the localization of CFTR at the level of the cellular membrane by expressing the CFTR cDNA carrying both F508del deletion combined with the neutralizing mutations. In the experimental systems used by the research team, that specific point mutations partially neutralized the impairment generated by the F508del genetic alteration. The results were obtained by measuring the localization of F508del mutation at the cell surface. The validated mutations were then introduced in the F508del mutated locus using a “second generation” CRISPR/Cas technology consisting of a nuclease free Cas combined with functional domains (deaminases). These systems, named “base editor” specifically modify the bases of the nucleotides without generating DNA double strand breaks as the original technology. The researchers used the base editors to introduce the “neutralizing” mutations proven to revert the F508del mutations in our experiments. The research team is moving forward with an additional financial support (FFC#2/2021) to identify new neutralizing mutation possibly editable with the “base editors” strategy.

Pubblications

• Maule G, et al. Gene Therapy for Cystic Fibrosis: Progress and Challenges of Genome Editing, International Journal of Molecular Sciences, Int J Mol Sci. 2020