Development of CRISPR/Cas9-based genome editing for the generation of allogeneic CAR-T cells

Abstract

Despite the remarkable clinical results, the autologous nature of CAR-T cell manufacturing process restricts the access of patients to this therapy. Therefore, one of the next frontiers in the field is to develop an allogeneic, 'off-the-shelf', CAR-T cell therapy. Initial studies have already provided a proof-of-concept of such "universal" therapy by knocking out the T cell receptor (TCR) from CAR-T cells using gene editing tools. Removal of TCR prevents the development of Graft versus Host Disease, an oftenly fatal complication following the allogeneic transplant of T cells. Removal of the human leukocyte antigen class I (HLA-I), on the other hand, helps to reduce the chances of graft rejection. Therefore, the aim of this project is to establish a methodology for the generation of "universal" anti-CD19 CAR-T cells by knocking out the genes TRAC (T Cell Receptor Alpha Constant) and B2M (²2-microgobulin) using CRISPR/Cas9-based genome editing. For this, we will transduce T cells with a lentiviral vector encoding an anti-CD19 CAR and gRNAs against the selected genes, followed by electroporation with Cas9 mRNA. The alloreactivity of the edited cells will be evaluated in coculture assays with allogeneic lymphocytes and the therapeutic efficiency will be assessed in coculture assays with lymphoma cells and in a murine model of Burkitt's lymphoma. This project addresses one of the goals of the Center for Cell-Based Therapy (CEPID project 013/08135-2). We believe that the obtained results will allow the development of a platform for production of allogeneic CAR-T cells ready-for-use, in addition to providing the necessary genetic editing knowledge to target other genes that limit the therapeutic efficiency of CAR-T cells. (AU)