NanoLuc reporters identify COL4A5 nonsense mutations susceptible to drug-induced stop codon readthrough

Area of Science:

• Genetics
• Molecular Biology
• Nephrology

Background:

• Alport syndrome is a genetic disorder affecting the kidneys, ears, and eyes.
• It results from pathogenic variants in collagen IV genes (COL4A3, COL4A4, COL4A5).
• Nonsense variants leading to premature termination codons (PTCs) truncate collagen IV chains, preventing basement membrane assembly.

Purpose of the Study:

• To evaluate the feasibility of premature termination codon (PTC) readthrough therapy for Alport syndrome.
• To identify specific COL4A5 nonsense variants amenable to readthrough.
• To assess the functional capacity of readthrough-generated collagen IV products.

Main Methods:

• Utilized NanoLuc reporter systems in transfected cells to monitor translation and assess readthrough product function.
• Conducted full-length NanoLuc assays on 49 COL4A5 nonsense variants to screen for drug susceptibility.
• Employed split-NanoLuc assays to evaluate the heterotrimerization of readthrough products with other collagen IV chains.
• Observed readthrough in kidney cells from a Col4a5 PTC mouse model.

Main Results:

• Eleven out of 49 tested COL4A5 nonsense variants were susceptible to PTC readthrough using various drugs.
• Readthrough products from five nonsense mutations could form heterotrimers with collagen α3(IV) and α4(IV) chains.
• PTC readthrough was successfully demonstrated in kidney cells of a relevant mouse model.

Conclusions:

• Premature termination codon (PTC) readthrough therapy is a potentially viable treatment strategy for a subset of Alport syndrome patients.
• The study identified specific COL4A5 variants that respond to readthrough, paving the way for targeted therapeutic development.
• Functional assessment of readthrough products confirms their potential to restore basement membrane integrity in Alport syndrome.