Gene therapy approaches for correction of EEC phenotype in the cornea

Ectrodactyly–ectodermal dysplasia–clefting syndrome (EEC) syndrome is an autosomal dominant disease caused by mutations in the TP63 gene. p63 is a transcription factor essential for ectodermal development, oocytes genetic quality control and epithelia regeneration. Mutations in TP63 are associated with several syndromes and disorders, characterized by features like ectodermal dysplasia, limb malformations, orofacial clefting, skin erosions, urogenital defects, and female infertility. EEC syndrome patients not only exhibit these symptoms, but they also develop progressive limbal stem cell deficiency (LSCD) in adult age, due to the defective ΔNp63α isoform, which is responsible for the maintenance of the proliferative status of basal keratinocytes and of limbal epithelial stem cells (LESCs). The exhaustion of LESCs and corneal epithelium thinning, worsened by dryness due to defective meibomian and lacrimal glands, results in corneal ulcerations, scarring, the development of a vascularized conjunctival pannus and eventually visual impairment. Since p63 is part of a complex biological network, the exact mechanism behind the pathogenesis of EEC associateed LSCD is still not completely understood. The mutated proteins are unable to correctly bind their targets, many of which are related to stemness maintenance. Furthermore, since p63 autoregulates through various feedback loops, the mutation affects the levels of the protein itself, leading to its accumulation in the cell. Currently, LSCD in EEC patients is delayed with conservative therapies, but the only existing cure is allogeneic limbal tissue transplantation, which has a high failure rate due to immune rejection and LSCD relapse. Transplantation of genetically modified autologous stem cells, like oral mucosal stem cells (OMESCs), could be an alternative strategy to reconstruct the cornea. An additive gene therapy approach has the advantage of being mutation independent, thus is suited for any EEC patient and even for other p63 related diseases. A self-inactivating (SIN) lentiviral vector, originally developed for the treatment of junctional epidermolysis bullosa (JEB), was modified to carry the transgene for ΔNp63α cDNA, fused to EGFP for research purposes. The internal promoter contains enhancer elements from K14, which can restrict the transgene expression to undifferentiated basal keratinocytes and is regulated by healthy ΔNp63α itself. This approach has showed to be able to increase the lifespan and clonogenic potential of EEC OMESCs in vitro, without signs of immortalization. Since mutated p63 proteins have a dominant negative effect on healthy p63, RNA silencing of the mutated allele is another interesting therapy. We are focusing on a transient approach, delivering to the cornea siRNAs or Gapmers, which have shown to be able to downregulate the mRNA levels of the mutated allele in OMESCs. This strategy could prevent LSCD progression in children and young adults, but requires lifelong adherence and has to be tailored for each mutation.