Fluorescence-Based Screen of HDR-mediated stem cells gene editing to treat Epidermolysis Bullosa Simplex

Epidermolysis Bullosa Simplex (EBS) is a severe blistering skin disorder caused by gene mutations that encode structural proteins essential for skin integrity, such as keratin 14. This rare autosomal dominant condition accounts for about 70% of all EB cases1. It results in skin fragility, leading to blisters and erosions from minor trauma, typically due to mutations in the KRT5 and KRT14 genes1. The severity of EBS can range from localized to widespread. Managing EBS daily is challenging for both patients and their caregivers. Current treatments mainly provide symptomatic relief2-5, as no definitive therapies exist. This study aims to develop a primary keratinocyte model suitable for gene expression studies of the KRT14 gene associated with EBS. Keratin 14 is an intermediate filament protein that provides structural support and mechanical resilience to keratinocytes in the basal layer of the epidermis. Using the CRISPR/Cas9 system, we will fuse the GFP coding sequence to KRT14, resulting in the constitutive expression of GFP-KRT14 fusion proteins under the control of the endogenous promoter in human WT and EBS-derived primary keratinocytes. This model allows for easy and rapid detection of gene editing events, as fluorescent keratin 14 can be easily detected through immunofluorescence and cytofluorimetric analysis once the eGFP sequence is correctly inserted into the KRT14 locus. We will use rAAV6 as a donor template and promote the HDR pathway. HDR gene editing depends on various factors, including the design of the gRNA, accessibility to the genomic locus, and the size of the donor template. Therefore, we propose establishing a cellular model to optimize a clinical-grade protocol for CRISPR/Cas-mediated knock-in (KIN) strategy in epidermal stem cells. This model is intended to support a future ex vivo gene therapy approach for EBS patients, aiming to replace the entire coding sequence of the KRT14 gene in the first exon of the KRT14 locus. Additionally, since HDR genome editing is inefficient in primary cells, we will investigate ways to enhance the HDR pathway using chemical and genetic modulation and evaluate the impact of this treatment on the stem cell compartment. This cellular model has the potential to serve as a platform for screening personalized gene editing approach and applications (such as base editing and prime editing for Krt14). At the heart of this approach is the promise of targeting all mutations responsible for KRT14-EBS mutations with unprecedented precision. This circumvents the need for multiple tailored treatments and overcoming the limitations of traditional therapies that often struggle to address the diversity of mutations in affected individuals.