Mentor Name: Ruby Sims

Cell therapy-based approaches including the generation of chimeric antigen receptor T cells (CAR T cells) provide additional treatment options for relapsed and/or refractory childhood cancers, showing promise for reducing the long-term side effects associated with existing pediatric oncology treatments. However, the manufacturing of therapeutic CAR T cells requires costly viral vector-based ex vivo modification, preventing their broader implementation. Lipid nanoparticles (LNPs) are a safe and highly effective method for delivery of nucleic acid cargos and have enabled non-viral, in vivo, transient engineering of CAR T cells in mice. To achieve non-viral integration of CD19 CAR in resting T cells, scalable ex vivo gene editing strategies must be improved. The CRISPR/Cas9 system enables site-specific insertion of a gene coding for a CD19 CAR construct at the TRAC locus, but its reliance on the cell cycle dependent homology directed repair (HDR) pathway remains a challenge for editing resting T cells in the G0 phase. Under the guidance of my mentor Dr. Ruby Sims, I seek to optimize an LNP based platform for the CRISPR/Cas9 mediated generation of CD19 CAR T cells. While data from our lab suggests that conjugation of anti-CD3 monoclonal antibodies to LNPs leads to improved transfection efficiency and activation of resting primary T-cells, I will explore the effects of LNP mediated T cell activation on CD19 CAR integration efficiency. Based on previous studies, we hypothesized that overactivation of CDK1, activated by cyclin B1, will promote activity of proteins involved with end resection, the critical step for biasing a cell towards HDR and away from NHEJ. Our preliminary data demonstrates the use of LNPs to transfect cells with mRNA coding for cyclin B1 and an EGFP reporter to Jurkats. Initially, we will apply anti-CD3 LNPs containing an mCherry reporter DNA donor template, Cas9 mRNA, TRAC targeting sgRNA, and cyclin-B1-EGFP mRNA to primary human T cells, quantifying overexpression of cyclin B1 via immunostaining and western blot, while exploring what, if any, effect it has on HDR integration at the TRAC locus. Furthermore, cyclin B1 mRNA will be assessed for its ability to allow gene insertion without the need for T-cell activation. Integration efficiencies and cell cycle/activation progression will be assessed via fluorescent antibody staining and flow cytometry. We hope that this work will contribute to more efficient generation of off-the-shelf CAR-T cell therapies, with potential application to safe in vivo CAR T cell generation, ultimately reducing costs and expanding access to these lifesaving treatments for pediatric oncology patients.