Combined gene and stem cell therapy approach to correct ocular defects in p63-related diseases

Background p63 gene is essential for the regeneration of adult epithelia and the maintenance of the proliferative status of basal keratinocytes. Mutations in p63 gene can cause at least five different syndromes (EEC, ADULT, AEC, LMS, and RHS) as well as two non-syndromic disorders (SHFM and NSCL). The three main phenotypic characteristics of p63-associated disorders are: (i) ectodermal dysplasia; (ii) split hand/foot malformation and (iii) orofacial clefting. The progressive keratopathy associated with limbal stem cell failure results in a vascularized pannus, leading to corneal clouding and visual impairment. In patients with bilateral limbal stem cells deficiency, transplantation of cultured autologous oral mucosal stem cells (OMESCs) sheets could represents an effective alternative to reconstruct the ocular surface. However, being genetic disorders, stem cells will have to be genetically modified. Aim of the study Allele specific silencing of mutated transcript mediated by siRNAs has yet been seen as potential therapeutic strategy in p63-mutated patients, in order to counterbalance the loss of stem cells (Barbaro et al., 2016). But the limits of siRNAs approach are that it is a transient and mutant-specific correction. So the ultimate outcome of my doctoral work has been to develop permanent gene therapy approaches to correct the genetic defect and to design a therapeutic strategy using the transplantation of this genetically modified stem cells. Methods A self-inactivating (SIN) lentiviral vector, with transgene expression (ΔNp63α) driven by cellular promoter with short-range enhancer activity (K14), was produced in order to restore the WT phenotype in mutated cells. It was evaluated if the behavior of transduced mutated cells was comparable with the WT. Luciferase reporter assay was performed to test the capability of p63, in its WT or mutated forms, to activate K14 promoter. Results We characterized OMESCs from patients with EEC syndrome and demonstrate a proof-of-principle to correct the underlying genetic defect by allele-specific silencing. It was designed a lentiviral vector similar to that used in junctional epidermolysis bullosa (JEB), modified with WT-ΔNp63α fused EGFP instead of laminin-5, and different cell lines were transduced. The signal of tagged EGFP is restricted in epithelial p63-expressing stem cells, demonstrating the possible use of this kind of vector in EEC syndrome therapy approaches based on genetic correction of oral mucosal keratinocytes. This approach, by acting on the amount of WT-p63 instead that mutated, has the great advantage of been mutation-independent, potentially useful not only in EEC syndrome but in all disease correlated with p63 mutations. In fact it was tested, by using luciferase assay, the activation of K14 promoter/enhancer by WT- or mutated-ΔNp63α. Results demonstrate that mutated-ΔNp63α are unable to activate the promoter, ability restored by adding increasing amount of WT-ΔNp63α. So it was tested the ability of this lentiviral vector to act in primary OMESCs. WT and R304Q transduced keratinocytes show an amount of ΔNp63α approximately 2-times higher than negative control, correlates with higher life-span (WT-OMESCs = 10-11 passages; transduced-WT-OMESCs = 12; R304Q-OMESCs = 5-6; transduced R304Q-OMESCs = 7) and CFE assay results. Discussion and conclusion Reported important results demonstrate that combined gene and stem cell therapy approaches for reconstructing the ocular surface represents an effective alternative for corneal blindness in mutated-p63 patients. It was demonstrated an improvement in the phenotype of R304Q cells transduced with our vector. Our hypothesis is that, using a lentiviral vector carrying ΔNp63α, whose expression is driven by K14 promoter/enhancer that is regulated by p63 itself, the increment of p63 would be not too high but presents an expression WT- stem cells like, recovering normal phenotype.

Lo scopo di questa ricerca è di correggere il difetto del gene p63 che comporta la perdita della vista in modo indipendente dalla mutazione. La strategia prescelta è una terapia additiva in cui viene impiegato un vettore lentivirale auto-inattivante (SIN) che esprime un transgene (WT ΔNp63α) sotto il controllo di un enhancer tessuto-specifico (K14). Questo vettore viene usato per ripristinare il fenotipo WT. Il potenziale di questa strategia è che potenzialmente può essere impiegata per migliorare il fenotipo di tutte le patologie correlate a p63. I nostri risultati mostrano che il comportamento e l’attività delle cellule mutate trasdotte possono esseri comparati a quelli del WT. Abbiamo inoltre osservato che le cellule WT mostrano un potenziale clonogenico maggiore rispetto al controllo negativo, senza comprometterne l’identità. Tuttora non ci sono cure disponibili per la perdita della vista causata da mutazioni in p63. La nostra ricerca potrebbe quindi condurre a nuovi approcci terapeutici basati sul trapianto autologo di cellule staminali geneticamente corrette. Inoltre la nostra strategia potrebbe essere in grado di curare un ampio spetto di patologie legate a p63 in modo mutazione indipendente.