Skin-targeted Cell Therapy for Recessive Dystrophic Epidermolysis Bullosa

Recessive dystrophic epidermolysis bullosa (RDEB) is a rare inherited mucocutaneous disease caused by mutations to the COL7A1 gene. Advancements in gene therapy have opened avenues for treatment by introducing a functional copy of the mutated gene in the patient’s cells. Viral vectors, gene editing nucleases, base editors, and prime editors are potential strategies for restoring expression of COL7A1. Lenti-, retro-, and herpes simplex virus (HSV) approaches are all being employed clinically. Ex vivo cell correction with integrating vectors (i.e. lenti- or retroviral) requires specialized facilities for engineering cells and these systems pose insertional mutagenic risks that are a consideration in the RDEB phenotype that has a predisposition to aggressive squamous cell carcinoma development. HSV-1 is a non-integrating vector that reduces genome toxicity related safety concerns but episomal nucleic acid could be lost over time. All of these viral vector systems mediate supranormal gene expression levels. In situ, locus specific correction is desirable as it retains the endogenous gene regulatory network and minimizes insertional mutagenesis. Here we will evaluate highly promising genome engineering and delivery approaches as part of a strategy to restore COL7A1 expression in cells ex vivo and in vivo. In the first aim, we will define and optimize evolved engineered Prime Assisted Site- Specific Integrase Gene Editing (eePASSIGE), a powerful addition to the genome engineering armamentarium. Polymeric nanoparticles (PNP) will be employed for the delivery of eePASSIGE nucleic acids that will install a full length COL7A1 cDNA into the endogenous locus ex vivo and in vivo. Mesenchymal stromal cells (MSC) that express ATP-binding cassette superfamily member (ABCB5) display homing and tissue repair properties and we will evaluate the ability of eePASSIGE corrected autologous ABCB5+ MSC to correct RDEB pathology in mice in vivo. In the second aim, we will develop a novel microfluidics-based epidermal poration device to deliver eePASSIGE PNPs topically to mice for in vivo, permanent COL7A1 gene correction. Both aims will then be synergized to assess the combinatorial potential of topical and cell-based therapy to ameliorate the cutaneous and internal disease manifestations toward filling a therapeutic gap in current treatment options. Our proposed experiments will develop a novel ex vivo and in vivo genome editing strategy that could be broadly applied across all RDEB patient cohorts and that will enhance overall efficiency and correct the full spectrum of the disease.