Cystic fibrosis: CFTR-gene editing may cure this genetic disease

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August 13, 2019 – A very significant research article by the title “Allele specific repair of splicing mutations in cystic fibrosis through AsCas12a genome editing” has just appeared in the open access Journal Nature Communications. This work provide hope to patients with cystic fibrosis (CF) that in the nearer future CF may be curable, not only treatable.

The lung of a cystic fibrosis (CF) patient.

But first things first. Cystic fibrosis (CF) is a lethal autosomal recessive inherited disorder with an approximate frequency of 1 in 2500 births. CF is linked to mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene, which encodes a chloride/bicarbonate channel expressed in the apical membrane of epithelial cells. The lack of ion conductance across the membrane of these cells leads to impaired ion and liquid homeostasis, generating a multi-organ disorder. The primary cause of mortality in CF patients is bacterial infections of the airways, provoking chronic lung disease and ultimately respiratory failure. Current CF treatments are not curative and limited to the reduction of clinical symptoms including intestinal-airway blockages and chronic bacterial infections. Recent therapeutic advances were obtained in CF treatments through the development of CFTR correctors and potentiators, which however target exclusively few types of mutations including the highly recurrent CFTRΔF508.

In search for a cure for CF, several gene therapy approaches have been explored, mostly based on CFTR cDNA gene addition through viral or non-viral vectors. Despite promising results obtained in the respiratory tract of animal models, and advancements in gene therapy clinical trials, curative goals were hampered mainly by low expression levels of the delivered CFTR.

In contrast to these classical gene addition strategies just mentioned, the correction of disease-causing mutated CFTR holds the promise to restore physiological levels of CFTR expression and function, i.e. leading to a true cure of the disease. Thus, recent advances in genome editing techniques, with the development of precise and efficient CRISPR-nucleases, have accelerated the progress of gene correction for genetic diseases, which may include CF. Following initial discovery of the Streptococcus pyogenes Cas9 (SpCas9), several additional CRISPR-nucleases have been discovered with different functional, mechanistic and structural features, expanding the genome editing tool-box. Among these, AsCas12a has been widely used for its natural very high specificity.

In the research presented here, a genome editing/correction strategy to repair 3272-26A>G (c.3140-26A>G) and 3849+10kbC>T (c.3718-2477C>T) CFTR mutations was developed. In fact, the 3242-26A>G is a point mutation that creates a new acceptor splice site causing the abnormal inclusion of 25 nucleotides within exon 20 of CFTR. The resulting mRNA contains a frameshift in CFTR, producing a premature termination codon and consequent expression of a truncated non-functional CFTR protein. The 3849+10kbC>T mutation creates a novel donor splice site inside intron 22 of the CFTR gene, leading to the insertion of the new cryptic exon of 84 nucleotides, which results in an in-frame stop codon and consequent production of a truncated non-functional CFTR protein. In the present study, the researchers harnessed the AsCas12a nuclease with a single CRISPR RNA (crRNA) to repair the CFTR 3272–26A>G and 3849+10kbC>T splicing defects in different cell types including primary CF patients’ airway epithelial cells and intestinal organoids. The genome editing strategy that they developed was highly specific, as demonstrated by a preserved second allele and complete absence of off-target cleavages. CFTR functional recovery by the AsCas12a single crRNA 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.
It remains to be seen if such gene correction strategies would eventually work with all the many mutations in CTFR that lead to the overt CF disease. It remains also to be seen if these gene correction therapy approaches are going to be free of unwanted or serious adverse effects.
See here a sequence on CF, its causes and underlying mechanisms. Note that are many more mutations in CFTR, such as those discussed in the present article that lead to the same physiological effect as discussed here:

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About the Author
Joseph Gut - thasso Ph.D.; Professor in Pharmacology and Toxicology. Senior expert in theragenomic and personalized medicine and individualized drug safety. Senior expert in pharmaco- and toxicogenetics. Senior expert in human safety of drugs, chemicals, environmental pollutants, and dietary ingredients.

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