Cystic Fibrosis (CF) is one of the most common genetic diseases worldwide. Although CF is considered a multi-organ disease, the lungs are the most affected organ. CF is caused by mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene, which encodes an ion channel present in secretory epithelial cells. CFTR protein transports water and, chloride and bicarbonate ions in and out of cells, creating a thin mucus layer that protects and lubricates several organs, including the lungs. Defective CFTR protein leads to impaired water and ions transportation, which causes thick mucus to build up in the lungs and promotes lung infection, ultimately leading to lung failure.
In the last 10 years, remarkable breakthroughs occurred in the CF field with the development of four CFTR modulators. These are small molecules that were designed to correct the defective CFTR protein, restoring its function. Since different mutations cause different defects in the protein, the modulators that have been developed so far are effective only in people carrying specific mutations. Currently, ~ 90% of the people with CF (PwCF) can benefit from these CFTR modulators. However, 10% of the PwCF still need targeted therapy.
Gene editing using CRISPR/Cas9 tools could potentially be a therapeutic option not only for this 10% of PwCF but also for all PwCF. CRISPR/Cas9 tool could potentially lead to a cure for CF.
How CRISPR/Cas9 works
CRISPR/Cas9, which is short for clustered regularly interspaced short palindromic repeats and CRISPR-associated protein 9, is part of the adaptative immune system of bacteria. When a virus infects bacteria, small viral DNA fragments are cleaved and inserted into the bacteria’s own DNA. If the same virus infects again, those DNA fragments are transcribed and guide Cas9 protein to recognize and cleave foreign nucleic acids, disabling the virus. Researchers adapted this defense mechanism of bacteria to edit human and other genomes in a programmable manner. As a genome editing tool, CRISPR/Cas9 system consists of two parts: the Cas9 nuclease and a guide RNA (gRNA). When delivered into cells, the gRNA binds to a specific DNA sequence, and the Cas9 cuts the two DNA strands at the targeted location, like the process in bacteria. The DNA cut triggers the cell’s own DNA repair machinery to insert or delete pieces of DNA, or to make specific changes by replacing the existing nucleotide bases with a customized DNA sequence.
CRISPR/Cas9 and Cystic Fibrosis
Since its discovery, CRISPR/Cas9 as a genome editing tool has revolutionized biomedical research, and there are already many studies showing its applicability to CF. The first study was published in 2013 in Cell Stem Cell and demonstrated the correction of mutant F508del allele using CRISPR/Cas9, fully restoring protein function in cultured intestinal stem cells of CF individuals.
F508del is the most common mutation on the CFTR gene representing ~ 70% of the alleles. This mutation causes the deletion of single amino acid, phenylalanine, from CFTR protein at position 508. Without this amino acid, the CFTR protein is not able to get a correct 3D shape and, consequently, the cell discards it. The combination of CFTR modulators Trikafta® (elexacaftor/tezacaftor/ivacaftor) works by enabling CFTR with F508del mutation to get a more correct shape and increase protein function. Although this drug combination is not perfect, the chloride that is able to flow through the channel is enough to improve CF symptoms, including lung function.
For other CFTR mutations, however, there are no CFTR modulators available. Such an example is nonsense mutations where no or very little protein is produced. As CFTR modulators work on the protein, PwCF carrying these types of mutations do not benefit from these drugs. Individuals carrying nonsense mutations represent ~ 12% of the CF community and for those CRISPR/Cas9 could potentially be an option. There are some studies demonstrating the correction of nonsense CFTR mutations, namely W1282X the second most common nonsense mutation in the CFTR gene. Such as the first study published back in 2013, these studies also show that correction of the W1282X allele restores CFTR mRNA and protein levels, and protein function.
Companies such as Editas Medicine, CRISPR Therapeutics, and Vertex Pharmaceutical are determined to discover and develop a CRISPR/Cas9 approach to potentially correct mutations in the CFTR gene. The Cystic Fibrosis Foundation launched a $500 Million program, called Path to a Cure, which prioritizes initiatives for individuals who do not respond to currently available drugs, including individuals carrying nonsense mutations. This program has three core strategies to address the root cause of the disease: repair CFTR protein, restore CFTR protein, and fix or replace the CFTR gene. Gene editing approaches are considered the most promising strategy to cure CF, as these approaches could potentially correct the faulty gene.
The Cystic Fibrosis Trust is also investing in a strategic research center (SRC) to explore the use of gene editing to fix CFTR mutations. Led by Professor Stephen Hart at the UCL Great Ormond Street Institute of Child Health, in London, this SRC also includes other principal investigators (PIs) in Cork (Ireland), and Paris (France). Their objectives are to 1) find the best gene-editing tool for the job, 2) find the most suitable method to deliver all the tools or the already-correct cells into the lungs, 3) investigate which type of lung cell to edit and whether the gene editing has restored the function of the CF protein within the cells, 4) test gene editing in animal models of CF.
The first CFTR modulator was approved in 2012 and 10 years and four modulators later ~ 90% of the PwCF benefit from targeted therapy. However, we cannot stop here as the goal is to find a treatment, and potentially a cure, for all CF individuals. CRISPR/Cas9 as a genome editing tool was also discovered 10 years ago and holds promise to cure cystic fibrosis.