Cystic fibrosis (CF) is autosomal recessive genetic disease that causes persistent lung infections and limits the ability to breath overtime. The disease is caused my mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene that cause the CFTR protein to become malfunctional. CFTR is an ion channel protein that transport chloride ions across the membranes of cells. Malfunction of the protein can lead to the production of thick mucus that obstructs airways and promotes infections. HHMI BioInteractive provides an excellent visualization of the disease state. CF is also called the invisible disease because it doesn't show on the outside, and yet it takes a huge toll on the lives of the people it affects.

Current treatments for CF patients are not curative and are limited to the reduction of clinical symptoms (intestinal-airway blockages and chronic bacterial infections). Recent therapeutic approaches target CFTR correctors and potentiators, but with limited success. Therefore, researchers at the University of Trento began to explore the use of genome editing to correct the mutation of CFTR that causes cystic fibrosis.

They hope to restore physiological levels of CFTR expression and function through this technique. To achieve the scientists applied genomic editing to mutations that alter the splicing of the CFTR gene (splicing is the mechanism of elimination of introns, ie the non-coding parts of the gene). In these mutations, the elimination of intron is sufficient to obtain gene repair and the correct definitive sequence. The technique is called "SpliceFix" because it fixes the gene and restores the protein production mechanism at the same time.

The new SpliceFix works to repair 3272-26A>G (c.3140-26A>G) and 3849+10kbC>T (c.3718-2477C>T) CFTR mutations. The researchers utilized the AsCas12a nuclease with a single CRISPR RNA (crRNA) to repair 3272-26A>G and 3849+10kbC>T. The strategy was validated in intestinal organoids derived from CF patients carrying the 3272-26A>G or the 3849+ 10kbC>T mutations, thus highlighting the power of this approach for the permanent correction of genetic diseases caused by deep intronic splicing mutations.

Giulia Maule, a doctoral student in Biomolecular sciences at the University of Trento and first author of the article, explained: “We demonstrated that our repair strategy works on patient-derived organoids and with a high level of precision: it targets only the mutated sequences, leaving non-mutated DNA untouched." The ability to permanently cure CF with this novel approach could be life changing for all CF patients and families.

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