Allele-Specific Prevention of Nonsense-Mediated Decay in Cystic Fibrosis Using Homology-Independent Genome Editing.

Nonsense-mediated decay (NMD) is a major pathogenic mechanism underlying a diversity of genetic disorders. Nonsense variants tend to lead to more severe disease phenotypes and are often difficult targets for small molecule therapeutic development as a result of insufficient protein production. The treatment of cystic fibrosis (CF), an autosomal recessive disease caused by mutations in the CFTR gene, exemplifies the challenge of therapeutically addressing nonsense mutations in human disease. Therapeutic development in CF has led to multiple, highly successful protein modulatory interventions, yet no targeted therapies have been approved for nonsense mutations. Here, we have designed a CRISPR-Cas9-based strategy for the targeted prevention of NMD of CFTR transcripts containing the second most common nonsense variant listed in CFTR2, W1282X. By introducing a deletion of the downstream genic region following the premature stop codon, we demonstrate significantly increased protein expression of this mutant variant. Notably, in combination with protein modulators, genome editing significantly increases the potentiated channel activity of W1282X-CFTR in human bronchial epithelial cells. Furthermore, we show how the outlined approach can be modified to permit allele-specific editing. The described approach can be extended to other late-occurring nonsense mutations in the CFTR gene or applied as a generalized approach for gene-specific prevention of NMD in disorders where a truncated protein product retains full or partial functionality.