Cystic Fibrosis (CF)

Cystic fibrosis (CF) is an autosomal recessive disease caused by defects in the CF transmembrane conductance regulator (CFTR) protein. With an estimated incidence in Europe of 0.737 per 10,000 births, it is the most common monogenic disease in Caucasian patients with autosomal recessive inheritance.

The CFTR protein is a cAMP-regulated chloride channel located in the membrane of epithelial cells. A mutation in the CFTR gene, which is one of the ATP-binding cassettes (ABC), located on chromosome 7, results in fewer CFTR channels or disrupts the functioning of these channels [2,3].

Although the CFTR protein acts primarily as a chloride channel, it has many other regulatory roles that can affect many metabolic pathways - for example, fatty acid metabolism. Studying these deregulations can lead to a better understanding of the disease. The molecular imaging of cellular metabolic pathways could also have a strong influence on the evaluation of the effectiveness of potential genetic therapies.

To date, more than 2100 different mutations in the CFTR gene have been described, but not all of them cause CF.

According to researchers, there are 6 classes of mutations [6,7] (Table 1). The most common mutation is of F508del (ΔF508) - about 90% of patients carry at least one copy of this allele - which is a three-nucleotide deletion at the 508th codon, which results in the deletion of a phenylalanine residue, followed by abnormal intracellular processing of the CFTR protein [8,9].

CF is a monogenetic disease but shows significant phenotypic variability - as evidenced by the wide spectrum of disease severity observed in patients with the same genotype. This may indicate that we should look for additional factors influencing the course of this disease.

The main organs affected by CF are the lungs. Defective CFTR reduces the release of Cl-ions from cells, what leads to the hyperactivation of epithelial sodium channels (ENaC). The high absorption of Na ​+ ​ions causes dehydration and thickening of the produced mucus.

Morbidity and mortality in CF are mainly caused by lung infections, bronchiectasis, small airway obstructions, and progressive respiratory failure. CF also affects other body systems and organs, due to epithelial cell dysfunction, such as the pancreas (malabsorption), sweat glands (heat shock), vas deferens (infertility), and liver (biliary cirrhosis).

In recent years, remarkable progress has been made in the treatment of CF. Multidisciplinary teams have applied innovative therapies targeting the basic defects of CF (such as lumacaftor, ivacaftor, tezacaftor, and elexacaftor). We have observed the development of drugs that facilitate the clearance of mucus from the lungs and reduceCF-associated infections. Nowadays, the accompanying pancreatic insufficiency and undernutrition are treated, which has proved more effective. This led to significant improvements in both clinical outcomes and quality of life of patients with CF. As a result of better treatment and increased survival rate in childhood, in the last 5 years, more adults than children have suffered from CF in countries with well-funded healthcare systems. In developed European countries, the number of adult CF patients is expected to increase by approximately 70% by 2025 [12,13].

However, there is a lot of room for new therapies - there are many mutations that still cannot be treated using pharmaceutical approaches.

There is a need for a causal therapy that can stop the disease at its source.

Currently, genetic tools, such as Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) that can correct the genome hold great promise for both CF and other genetic diseases. Research is promising and it seems to be a matter of time before technical hurdles will be overcome and effective CF therapy based on genetic editing applied.

Recessive monogenic diseases such as cystic fibrosis are optimal targets for therapy through genetic complementation. When CFTR modulators work on selected mutations, genetic interventions can target any alterations underlying the disease and potentially provide permanent disease resolution.

One of the most popular gene-editing methods is the use of CRISPR/Cas9. Due to the simplicity in target designing, high specificity, and low cost, it seems to be an appropriate tool for the treatment of CF.

In this article, we outline and characterize CRISPR/Cas9-based gene-editing technologies for CF modeling and potential therapy. We wish to highlight the role of human in vitro lung models derived from human-induced pluripotent stem cells (hiPSCs) as a resource for CF research. Since providing genome-engineering tools in vivo is still difficult but crucial, we present the latest research and methods.