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Abstract: FR-OR32

Development of a Personalized Medicine Platform for Nonsense Readthrough Therapy in Alport Syndrome

Session Information

Category: Glomerular Diseases

  • 1201 Glomerular Diseases: Fibrosis and Extracellular Matrix

Authors

  • Omachi, Kohei, Washington University in Saint Louis, Saint Louis, Missouri, United States
  • Kai, Hirofumi, Kumamoto University Graduate School of Pharmaceutical Sciences, Kumamoto, Japan, Kumamoto, Kumamoto, Japan
  • Miner, Jeffrey H., Washington University in Saint Louis, Saint Louis, Missouri, United States
Background

Alport syndrome (AS) is characterized by glomerular basement membrane (GBM) abnormalities leading to progressive glomerulosclerosis. Mutations in the COL4A3, COL4A4 or COL4A5 genes encoding type IV collagen α3α4α5 cause AS. Nonsense mutations resulting in premature termination codons (PTCs) account for about 6% of AS cases. Type IV collagen chains have a C-terminal NC1 domain, which is essential for assembly of heterotrimers inside cells and for network formation in the GBM. Truncated α3, α4, α5 chains without an intact NC1 domain due to PTCs cannot assemble in the GBM. Therefore, achieving full-length protein expression is a potential therapy for AS due to nonsense mutations. Small compound-based nonsense readthrough therapy has been well studied in other genetic diseases, but whether nonsense readthrough therapy is applicable to Alport syndrome is unexplored.

Methods

To investigate the feasibility of PTC readthrough in AS, we made a C-terminal NanoLuc-fusion COL4A5 reporter cDNA to monitor full-length translation. The full-length COL4A5-NLuc produces luminescence, but truncated forms do not. To screen for COL4A5 nonsense mutants susceptible to PTC readthrough therapy, we introduced 49 nonsense mutations found in X-linked AS patients into the COL4A5-NLuc gene. We transfected these individually into HEK293 cells and measured luminescence in the presence of Geneticin (G418), which is known to have high readthrough activity.

Results

The COL4A5-NLuc gene produced luminescence when wild type, but not when carrying COL4A5 nonsense mutations. Among 49 nonsense mutants, we found that 11 were susceptible to PTC readthrough. The efficacy of readthrough was higher for UGA nonsense codons than for UAG and UAA. Gentamicin also induced readthrough of these same 11 PTCs.

Conclusion

We found 11 nonsense mutations in COL4A5 susceptible to PTC readthrough drugs. This luciferase-based COL4A5 translation reporter system will contribute to the development of PTC readthrough therapy in a personalized medicine approach to treating AS.

Funding

  • NIDDK Support