Antisense oligonucleotides-mediated rescue of the AMELX splicing defects.

BACKGROUND/PURPOSE: Amelogenesis imperfecta (AI) is a group of rare, inherited disorders characterized by abnormal enamel formation. While identifying the genes and mutations that cause the disease is important, understanding its molecular pathogenesis is essential to developing precision and personalized medicine, which aim to stop disease onset, slow its progression, and provide a range of treatment options. Previously, we identified mutations that affect the conserved alternative splicing of the AMELX gene, leading to the inclusion of normally skipped exon 4 and resulting in a characteristic AI phenotype. MATERIALS AND METHODS: HEK293 cells were grown on cell culture dishes for an in vitro splicing assay, and transiently transfected with the wild-type, c.120T>C, or c.143T>C AMELX expression vector. Morpholino antisense oligonucleotides (ASO) were designed and tested for correction of the altered splicing pattern. The test was performed with ASO concentrations of 1, 2 and 4 μM. Total RNA was isolated for splicing analysis, and the cells and culture media were harvested for Western blot analyses. RESULTS: The altered splicing pattern was corrected by applying antisense oligonucleotides in vitro. The exon 4-included mutant mRNA was successfully eliminated, and the expression pattern of AMELX protein was restored. CONCLUSION: To the best of our knowledge, this is the first report to use ASO to silence mutant transcripts to correct splicing mutations confirmed to be disease-causing. This study represents a small step towards genetic interventions for AI and may serve as a basis for regulating the expression of mutant transcripts in developing teeth for future human studies.