The researchers showed that the corrected CFTR gene was functional and working normally in mini-intestines called organoids, which had been derived from CF patients and grown in the lab.
The study, “Allele specific repair of splicing mutations in cystic fibrosis through AsCas12a genome editing,” was published in the journal Nature Communications.
The gene editing tool CRISPR-Cas9 — a strategy that allows the correction of genomic defects — is considered one of the most promising approaches to correct the mutations in the CFTR gene. However, current attempts to correct these mutations have been hampered by the ineffective delivery of a normal CFTR gene.
In the study, researchers at the Trento University, in Italy, and Katholieke Universiteit (KU) Leuven, in Belgium, used a more precise CRISPR tool to correct permanently two CFTR gene mutations that block the production of a functional CFTR protein.
“We have devised a genome editing strategy based on Crispr-Cas to permanently remove two different mutations that cause the disease. Crispr-Cas works like a genomic scalpel to cut out the mutated elements with extreme accuracy,” Giulia Maule, doctoral student at Trento University and the study’s first author, said in a news release.
The team first tested its strategy in cells carrying either the normal (wild-type) or mutated versions of the CFTR gene. The CRISPR gene editing tool was able to restore the levels of normal CFTR mRNA molecules (the templates that cells use to make proteins) by more than 60% in a highly specific manner.
Next, the researchers investigated whether their molecular tool could be used in CF patients’ cells. For this, they used lung cells and mini-intestines that were grown from the intestinal epithelial cells of CF patients.
Results showed that the gene editing tool corrected the aberrant CFTR gene in both models, and recovered the function of the CFTR protein. Moreover, after 14 days, patient-derived organoids increased by 2.5 times their area, indicating that the CFTR channels were working normally.
“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,” said Maule.
“Instead of animal models, we have used organoids that we developed from the patients’ cells, a choice that allowed us to verify the efficacy of the molecular strategy in a context that is very similar to that of the patients with cystic fibrosis,” Maule added.
The researchers called their gene editing tool “SpliceFix,” as it can fix the CFTR gene and restore CFTR protein production.
Currently, patients with CF rely on the use of CFTR modulators that target the CFTR protein but have no effect on genetic defects. However, these therapies are currently limited and come with side effects, the researchers noted.
“This explains the urgent need for a permanent correction of physiologic levels of CFTR, potentially reachable with the genome editing approach described in our study,” the researchers concluded.
The team believes its gene editing tool can be applicable to other genetic defects in CF, and potentially to other genetic diseases.