Antisense Oligonucleotides-Mediated Rescue of the AMELX Splicing Defects

Objectives: The purpose of this study was to use antisense oligonucleotide (ASO) to restore the abnormal splicing pattern caused by mutations affecting the conserved alternative splicing of the AMELX gene.
Methods: The AMELX expression vector including exon 2 through exon 7 of the AMELX gene was cloned and PCR mutagenesis was performed to introduce the identified mutations: the silent mutation (NM_182680.1: c.120T>C) and missense mutation (c.143T>C). In vitro splicing assay was performed with transient transfection of HEK293 cells. ASO was applied at concentrations of 1x, 2x, and 4x, and the splicing pattern was analyzed. AMELX expression was tested in cell lysate and culture media by Western blot analysis.
Results: The altered splicing pattern was corrected by applying ASO at 1x concentration for the c.120T>C mutation and 2x concentration for the c.143T>C mutation. The protein expression pattern of AMELX was also restored.
Conclusions: We developed and applied an ASO-mediated strategy to rescue the AMELX splicing defects causing amelogenesis imperfecta (AI), and the exon 4-containing mutant mRNA was successfully eliminated; therefore, the expressed and secreted AMELX pattern was restored. To the best of our knowledge, this is the first attempt at gene therapy for AI, and it will serve as a basis for future human studies.