Point mutation in P63 leads to limbal stem cell deficiency that is rescued by a small molecular weight compound

Abstract

Purpose : The pathogenesis of genetic limbal stem cell deficiency (LSCD) is not well understood and therapeutic options are limited due to gap of knowledge and lack of reliable animal models. We therefore aimed to develop an in-vivo mouse model of genetically induced LSCD, that allows us to study the disease etiology and explore new therapeutic strategies.

Methods : Clinical diagnosis and genetic analysis was performed for LSCD patients to identify disease-causing mutations. A conditional mouse model carrying a mutation specifically expressed in LSCD patients was established. Abnormalities in the limbus, cornea and Meibomian glands were analyzed by microscopy, histology and immunofluorescent staining of wholemount and tissue sections. In addition, the abnormal molecular pathways were exposed by RNA-sequencing. Limbal stem cell clonal dynamics were uncovered by quantitative lineage tracing and the therapeutic potential of the small molecular weight compound PRIMA-1^MET was examined.

Results : P63^L514F was discovered as a new LSCD-causing mutation. We established a new conditional P63^+/L514F mouse strain that exhibits mild neovascularization that aggravates with age and eventually leads to loss of corneal transparency. Histological and immunofluorescent staining analyses revealed thickening of the corneal epithelium, abnormal presence of goblet, conjunctival and immune cells in the corneal center. Genetic lineage tracing showed abnormal size, orientation and survival of limbal stem cell-derived clones in mutant, in line with impaired wound repair. RNA sequencing analysis confirmed an enrichment of mucus secretion pathways, vascularization, and immune response. Finally, systemic treatment with PRIMA-1^MET significantly inhibited the development of the LSCD phenotype and improved wound healing response.

Conclusions : P63 plays a crucial role in corneal morphogenesis and adult LSC maintenance. The new P63^+/L514F strain is a useful model studying LSCD pathogenesis. Remarkably, PRIMA-1^MET has a promising therapeutic potential for the treatment of P63-related LSCD. Altogether, this study paves the way for further investigation of PRIMA-1^MET in clinical trials.

This abstract was presented at the 2023 ARVO Annual Meeting, held in New Orleans, LA, April 23-27, 2023.