Efficacy and safety of lentiviral vector for the treatment of p63-related visual morbidities

The Ectrodactyly-Ectodermal dysplasia-Clefting (EEC) syndrome is an autosomal dominant inherited disease due to mutations in the p63 gene, located on chromosome 3q27. This syndrome causes split-hand and foot malformations, and ocular morbidity, the last one being mostly caused by limbal stem cell deficiency (LSCD). So, mutations in p63 gene are the putative responsible for the thinning of the corneal epithelium and the progressive exhaustion of limbal stem cells. Since the pathogenetic mechanisms are due to the presence of mutation in p63 gene, it is possible to correct, at least in part, some of the defects by means of gene therapy approaches. RNA interference (RNAi) is one of the mechanisms under study; it requires the interference of gene expression through the introduction of small RNA fragments that are able to antagonize the mRNA, thanks to their complementary sequence. The RNA:RNA pairing is recognised by the cellular defense mechanisms and it will be degraded, by sequestering the mRNA that will not be translated into a protein. Thus, in the case of EEC syndrome affected patients, siRNA molecules can be delivered in the young patients’ limbal stem cells to downregulate the mutated allele of p63, thus preventing the progressive exhaustion of stem cells in the corneal epithelium. This approach, however, requires continuous administration of the drug throughout the patient’s life, with possible associated side effects. In addition, it cannot be applied for adult patients who no longer have the limbal stem cells, which, because of their continuous activity to substitute the corneal epithelium, will easily get exhausted. New therapeutic solutions are therefore necessary and deserve to be explored. In previous experiments, an additive gene therapy for the correction of the EEC syndrome genetic defect was evaluated. It was observed that the viral particles were able to transduce both HEK 293T cells and OMESCs carrying the R304Q mutations, derived from oral mucosa of EEC patients; the viral particle were able to integrate and provide ΔNp63α expression, and mutated cells showed increase life-span for one more passage compared to the negative control. In my work, I will dive deep into the details of p63 interaction with the mutant isoforms through BRET experiments and evaluation at the confocal microscope of the nuclear or cytoplasmic presence of p63 in the cells. To assess the efficiency of transduced R304Q-OMESCs cells to provide a stratified epithelium, organotypic cultures over human keratoplasty lenticules as a scaffold are produced and analyzed at both fluorescence and confocal microscope. Then, analyses on the integration sites help us understand the safety of such a vector compared to other lentiviral vectors used in clinical trials.