摘要：SummaryAlport syndrome, a disease of kidney, ear, and eye, is caused by pathogenic variants in theCOL4A3,COL4A4, orCOL4A5genes encoding collagen α3α4α5(IV) of basement membranes. Collagen IV chains that are truncated due to nonsense variants/premature termination codons (PTCs) cannot assemble into heterotrimers or incorporate into basement membranes. To investigate the feasibility of PTC readthrough therapy for Alport syndrome, we utilized two NanoLuc reporters in transfected cells: full-length for monitoring translation, and a split version for assessing readthrough product function. Full-length assays of 49COL4A5nonsense variants identified eleven as susceptible to PTC readthrough using various readthrough drugs. In split-NanoLuc assays, the predicted missense α5(IV) readthrough products of five nonsense mutations could heterotrimerize with α3(IV) and α4(IV). Readthrough was also observed in kidney cells from an engineeredCol4a5PTC mouse model. These results suggest that readthrough therapy is a feasible approach for a fraction of patients with Alport syndrome.Graphical abstractDisplay OmittedHighlights•NanoLuc fusion constructs identifiedCOL4A5mutants susceptible to PTC readthrough•Readthrough enhancer and “designer” compounds promoted PTC readthrough•Split-NanoLuc fusion constructs identified functional missense readthrough products•CulturedCol4a5nonsense mutant mouse kidney cells were susceptible to readthroughBiochemistry; Molecular biology; Molecular medicine
