Allele-specific silencing as treatment for p63-related disorders

Ectrodactyly-ectodermal dysplasia-clefting (EEC) syndrome is an autosomal dominant disorder caused by mutations in the TP63 gene, which encodes the transcription factor p63. This protein is critical for ectodermal development, epithelial regeneration and oocyte quality control. EEC syndrome is associated with a spectrum of clinical features, including ectodermal dysplasia, limb malformations, orofacial clefting, and progressive limbal stem cell deficiency (LSCD), which leads to severe visual impairment in adulthood. Current treatments are limited to palliative care, emphasizing the need for novel therapeutic strategies. In this study, we explored an innovative allele-specific silencing therapy targeting mutant p63 alleles. Our laboratory previously identified a small interfering RNA (siRNAα) capable of specifically silencing the mutant TP63 R279H allele in Human Embryonic Kidney Cells (HEK-293T) and Oral Mucosal Epithelial Stem Cells (OMESCs) without affecting wild-type allele expression. This approach extended OMESCs lifespan, delayed stem cell differentiation, and reduced abnormal proliferation in vitro. However, the successful therapeutic application of siRNAs require efficient delivery systems to facilitate their entry into Limbal Stem Cells (LSCs). Several eye drop formulations, combining Lipofectamine with siRNAα were tested, but all exhibited cytotoxicity after prolonged use. Moreover, eye drop formulations interfered with the Lipofectamine-mediated transfection process. To address these challenges, we evaluated micelles and lipoplexes as siRNA delivery vehicles in R279H-OMESCs. Micelles failed to achieve effective gene silencing. In contrast, lipoplexes successfully mediated siRNA-induced gene silencing in a transfection duration- and concentration-dependent manner. Additionally, we explored the use of GapmeRs, short antisense DNA oligonucleotides, as an alternative allele-specific silencing approach to circumvent the need for a transfection agent. GapmeRs designed for the R279H and R279C mutations selectively suppressed mutant alleles without affecting wild-type allele expression, while those targeting the R304Q mutation failed to induce gene silencing. These results highlight the potential of allele-specific therapies, particularly siRNA and GapmeR-based approaches, for treating EEC syndrome and mitigating its progressive ocular complications.