Abstract Recessive dystrophic epidermolysis bullosa (RDEB) is a prototypical genodermatosis caused by biallelic loss- of-function mutations of COL7A1. These mutations lead to a lack of type VII collagen (C7) in the skin and mucosal membranes, resulting in a complex phenotype of blistering, fibrosis, pseudosyndactyly, joint contractures, esophageal strictures, corneal abrasions, malnutrition, autoimmunity, anemia, and squamous cell carcinoma. Despite tremendous efforts over the last decade to establish curative measures for this severe and potentially fatal disorder, there are as yet no therapies that reliably supply C7 protein to the multiple sites affected by generalized severe RDEB. To address this, we propose to gain a more mechanistic understanding of how to restore the integrity of COL7A1 without causing collateral damage to the rest of the genome, and of how the specialized tropism of cells works to deliver intact, functional C7 throughout the body. In order to accomplish these goals and to overcome the limitations of current gene and cell therapies, we will investigate the following questions: [i] Is base editing superior to CRISPR/Cas9-editing for correction of COL7A1 mutations? Because base editing does not cause double-strand breaks in the way that classic gene editing with DNA nucleases does, it avoids genotoxic stress. [ii] Are skin-specialized cells, such as ABCB5+ mesenchymal stromal/stem cells (MSCs), superior to alternative sources of MSCs in expression of C7 levels adequate for cross-correction of C7 deficiency in RDEB? We will evaluate skin-specific stromal cells, such as mesenchymal stromal cells expressing ATP-binding cassette sub-family B member 5 (ABCB5+) surface protein, derived directly from skin or indirectly from patient-specific induced pluripotent stem cells, which have had COL7A1 restored to function with base editing. [iii] Do COL7A1-edited human ABCB5+ MSCs mediate wound healing in a preclinical murine model of RDEB? Using our murine model of RDEB that accepts human xenografts, we will quantify the value of base editing-corrected ABCB5+ MSCs and induced pluripotent stem cell-derived MSCs. We propose to define the conditions conducive to wound healing in this severe blistering genodermatosis by using powerful tools for studying and manipulating the information bases of biological systems (i.e., programmable deaminases for base editing-mediated gene therapy; induced cell lineage conversion; and skin tropism). We will aim for personalized cell therapy for individuals with generalized severe RDEB, with the idea that our findings may provide insights into ways to manage other genodermatoses, as well as treatment of mucocutaneous ulcers, and chemical and thermal burns. Our proposal is equally motived by wanting a better understanding of the biological mechanisms in injured skin and by needing to improve the lives of people with RDEB through reducing the risks and maximizing the benefits of potential novel gene and cell therapies.

Project Narrative In response to the need for effective treatments for severe genetic disorders, we are exploring gene and cell therapies in a skin-blistering disease—recessive dystrophic epidermolysis bullosa (RDEB)—caused by errors in a single gene. Our laboratories combine areas of expertise specific to this project: extensive research into RDEB, production of unique mouse models, a long history of cellular therapy, and state-of-the art gene editing technologies in operation. We hope to identify which of the current gene-editing technologies are safest and most effective to use in this disorder, to identify the optimal cells for correction and skin repair, and then to test the corrected optimal cells in a mouse model we have developed (that has RDEB and will accept human cells) to see if we can improve skin healing with this therapy.