HSV-1 base editing: exploring novel therapeutic frontiers for incurable p63-related disorders

Mutations in the TP63 gene cause a heterogeneous group of autosomal dominant disorders, commonly referred to as p63-related diseases. These conditions can severely affect multiple tissues, including the skin and its appendages, mucous membranes, teeth, and the ocular surface. Among the most debilitating complications are those involving the eye, which may lead to limbal stem cell deficiency (LSCD), a severe condition characterized by impaired corneal regeneration, corneal conjunctivalization, and progressive vision loss. Currently, no definitive curative strategy is available for the treatment of corneal alterations in p63-related disorders. The gold standard for restoring the corneal surface in unilateral LSCD patients is corneal transplantation; however, this strategy is not suitable for total, bilateral cases, mainly due to high risk of immune rejection and the need of long-term systemic immunosuppression, which affect long-term graft survival. Since the cornea represents an ideal tissue model for gene therapy and genome editing, this thesis project aims to develop a novel base-editing approach, through an innovative delivery system: HSV-1 derived amplicon vector. This platform was selected primarily for its high cargo capacity (up to 150 kb) and its non-integrative nature, making it suitable for delivering the base-editing machinery. Our primary goal is the targeted correction of the pathogenic mutation R304Q in the TP63 gene, responsible for EEC syndrome. To this purpose, adenine base editor (ABE) platform will be validated in an in vitro model, generated using a third-generation lentiviral vector to stably integrate the hTP63 R304Q fused to an EGFP reporter gene. The thesis will focus on assessing base editing efficacy in restoring the wild-type phenotype in this in vitro model, with the future goal of translating it in an ex vivo OMESC model to assess its clinical potential for p63-related corneal diseases. Although this study focuses on EEC syndrome, the proposed strategy could be extended to other p63-related disorders, such as AEC, RHS, LMS, and ADULT syndromes, that share similar genetic mutations and stem cell dysfunctions. This platform may thus offer broader therapeutic potential for multiple currently untreatable TP63-associated conditions.