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Prime editing allows tackling Cystic Fibrosis at its genetic roots

By: Mattijs Bulcaen, Marianne Carlon

A multidisciplinary team with researchers from KU Leuven (Flanders), Université Paris Cité (France), and the University of Trento (Italy) was able to precisely correct mutations that cause a life-threatening lung disease known as Cystic Fibrosis (CF). In May, their manuscript entitled “Prime editing functionally corrects Cystic Fibrosis-causing mutations in human organoids and patient airway epithelial cells” was published in Cell Reports Medicine.

The researchers leveraged prime editing, one of the most recent and advanced CRISPR-derived technologies, to restore mutations in the CFTR gene. Central in the development and testing of new prime editing approaches were the KUL-located patient-derived organoid and airway epithelial cell biobanks, but also the computing resources provided by the VSC for detailed analysis of DNA editing on the intended target site and potential off-target sites in the genome.

CF is Europe’s most common life-threatening inherited disease and has an estimated 160.000 patient population worldwide. Perhaps the archetype of an autosomal, recessive disorder, CF arises when both alleles of the CFTR gene contain CF-causing mutations. CFTR codes for the Cystic Fibrosis Transmembrane conductance Regulator (CFTR) protein, an essential ion channel present in many organs of the body. When the channel is absent or dysfunctional, chloride transport over epithelial layers is disrupted, leading to dehydration of the surfaces. In the lungs, this leads to the accumulation of thick mucus layers causing ineffective clearance of pathogens, chronic infection, inflammation, difficulty breathing, and a severe and progressive disease.

The computing resources, as well as the excellent documentation and support provided by the Flemish Supercomputer played an invaluable role in providing proof of concept for achieving precise DNA correction through the developed prime editing approaches and establishing a genome-wide safety profile for this novel technology. Also in our follow-up research, the VSC will maintain its pivotal role as key facilitator of discovery and genomic profiling.”

Since CF is a completely monogenic disease, gene therapy or gene editing therapy could be a promising therapeutic avenue. To investigate the feasibility of such therapy, researchers at KU Leuven tested different prime editing approaches in HEK293T cell lines, later moved to 16HBE cells and next to patient-derived intestinal organoids and in vitro differentiated airway epithelia. In every model, correction was the default readout, but every time precise DNA correction was observed, it was followed by a restoration of the CFTR protein and by rescued CFTR function.

Although prime editing has a much higher fidelity compared to other CRISPR approaches, unintended modifications at similar sites in the genome remain a main concern. To evaluate this, GUIDE-seq was used to pinpoint risk sites. To find these, cells were treated with a cleaving CRISPR enzyme and with barcodes that can integrate at the cut-sites. Through NGS, these barcodes were retrieved, thereby providing coordinates and frequencies for off-target editing. Next, Illumina NovaSeq was used to deep sequence the identified risk sites in organoids exposed for two weeks to high levels of the prime editing machinery. The fact that after these two weeks no indications of off-target activity could be found underscores the potential of prime editing as a future therapeutic therapy for CF, but by expansion also other severe genetic diseases.


Read the full publication in the Cell Reports Medicine here


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