Precision Editing of Cyclophilin A Generates Cyclosporine and Voclosporin Resistant Cellular Therapies

Patients who are recipients of allogeneic transplants or have underlying autoimmune disease require immune suppression, often with calcineurin inhibitors (CNI). There is an expanding repertoire of immune effector cell (IEC) therapies, including CD19 CAR-T cells and viral-specific T cells (VSTs), deployed in these patients; however, ongoing CNI therapy may be detrimental to IEC function. We thus developed a CRISPR/Cas9-based approach to engineer dual CNI [cyclosporine (CsA) and voclosporin (VCS)] resistant IEC therapies by targetingPPIA(encoding cyclophilin A - CypA), a critical binding partner for both drugs. Because CypA has several homeostatic functions in T cells, a complete CypA knock-out could impair cell viability. To avoid this, we edited the last exon of thePPIAgene, corresponding to the C-terminus of CypA, selectively disrupting amino acids that mediate CsA/VCS-based inhibition, while leaving the majority of CypA intact. Unlike an edit in an upstream exon, which was detrimental to cell survival and rapidly selected out, C-terminal editing was stable throughout expansion and preserved CypA protein expression. This edit was then introduced into two types of IECs. Edited CD19 CAR-T cells retainedin vitroeffector function in the presence of CsA/VCS, including preserved proliferation, target cell killing, and cytokine production. Edited CMV-specific T cells demonstrated antigen-specific proliferation and cytokine production in the presence of CsA/VCS. This report of site-specific CypA modification offers a promising avenue for developing next-generation IECs that should function effectively in patients receiving CsA/VCS and thus expand applications for adoptive cell therapies in multiple clinical settings.