Multiplex Engineered Human Lymphocytes for Therapeutic Protein Delivery

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Abstract. Recessive dystrophic epidermolysis bullosa (RDEB) is a severe autosomal recessive disease caused by collagen type VIIa (COL7A1) gene mutations. RDEB is characterized by absent/defective COL7A1 (C7) protein deposition causing severe blistering, mucosal tissue damage, and aggressive squamous cell carcinoma. The University of Minnesota serves as a leading treatment referral center for this inherited disorder. Palliative care is non-curative and cellular therapy options include autologous or allogeneic local and/or systemic infusion of keratinocytes, fibroblasts, mesenchymal stromal cells (MSC), or hematopoietic stem/progenitor cells (HSPC). None of these currently employed treatment options resolve the full pathological spectrum of RDEB. Active wound areas persist, and mucosal disease remains highly refractory to intervention contributing to significant morbidity. Keratinocytes and fibroblasts, the primary C7 producing cells, show limited migration and persistence following localized injection. MSC and HSPC have broad circulatory potential, however, they produce comparatively low levels of C7 and residence in the skin or mucosa is not well established. Thus, it is essential to develop more efficacious cellular therapies capable of accessing skin and mucocutaneous tissues. γδ T cells are abundant within skin and mucosa, and due to their MHC-unrestricted nature are compatible with allogeneic transfer, however they do not naturally produce C7. Our innovative approach will employ precision genome modification using CRISPR/Cas9 to engineer γδ T cells to produce high levels of endogenous C7. We hypothesize that the tissue migratory properties of γδ T cells—particularly to the skin and mucosa—as well as their demonstrated allo-compatibility, make them uniquely suited for therapeutic delivery of C7 protein. In Aim 1 we will define a genome engineering strategy to confer high C7 expression, enhanced skin homing, and reduced inflammatory/cytotoxic capacity to primary human γδ T cells. In Aim 2 we will evaluate the ability of engineered allogeneic γδ T cells to home to skin and mucosa, deposit C7, and ameliorate pathology in an immunodeficient mouse model of RDEB. Further, we will test the effect of zoledronate induced in vivo expansion of the engineered Vγ9Vδ2 T cell subset on therapeutic efficacy. Our approach is a highly novel and innovative allogeneic strategy designed to address key limitations of current cellular therapies for RDEB. The application of CRISPR/Cas9 in γδ T cells represents a novel engineering and protein delivery strategy with translational potential for RDEB, other inherited mucocutaneous disorders, and a multitude of diverse disorders treated by cell/stem cell transplant.