DEVELOPMENT OF CRISPR/CAS9-BASED GENOME EDITING PLATFORM FOR THE GENERATION OF ALLOGENEIC CAR-T CELLS

Abstract

<h3>Background</h3> Despite its remarkable clinical success, the autologous nature of chimeric antigen receptor (CAR)-T cell therapy restricts the access of patients to this treatment. Thus, one of the next frontiers in the field is to develop an allogeneic, ‘off-the-shelf' therapy. This can be achieved by knocking out the T cell receptor (TCR) from CAR-T cells using genome editing tools to prevent the development of graft versus host disease, an often fatal complication following the allogeneic transplant of T cells. In this study, we establish a methodology for depleting the TCR by knocking out the TRAC gene using CRISPR/Cas9 genome editing. <h3>Methods</h3> We tested two guide RNAs (gRNAs) sequences against the TRAC gene (TRAC1 and TRAC2) which were cloned into lentiviral vectors upstream the anti-CD19 CAR gene. Lentiviral particles were used to transduce Jurkat cells. These cells were used as a model for TCR depletion after electroporation of a plasmid for transient expression of a high-fidelity Cas9. <h3>Results</h3> After transduction, CAR+ Jurkat cells were enriched by magnetic selection to obtain homogeneous expression of CAR and TRAC gRNAs. Flow cytometry analysis after selection demonstrated that 96.3 and 97.7% of cells (TRAC1 and TRAC2, respectively) expressed CAR on the surface. Next, cells were electroporated with a plasmid encoding Cas9 and GFP using two different media: Solution V and Opti-MEM. After 24h of electroporation in Solution V, CAR-T cells displayed high viability (∼90%) regardless of the presence of Cas9-GFP plasmid. In Opti-MEM medium, however, electroporation induced high cell death rates (12.1-16.0% viability). Furthermore, the frequency of GFP+ cells after electroporation in Opti-MEM, was 1.6-fold (TRAC1) and 1.9-fold (TRAC2) lower compared to Solution V. To evaluate the efficiency of gRNAs in depleting the TCR, electroporated cells in Solution V were analyzed after 1, 3 and 8 days for CD3 expression. Notably, TRAC gRNA 1 led to a remarkable reduction in the frequency of CD3+ cells (from 91.6 to 7.41%). In contrast, this reduction was less pronounced for TRAC gRNA 2 (decrease of 21.8%). <h3>Conclusion</h3> These data demonstrate the feasibility of TCR depletion by knocking out the TRAC gene using CRISPR/Cas9 and establish the best gRNA sequence for this purpose, paving the groundwork for the establishment of a platform to produce ‘off-the-shelf' allogeneic CAR-T cells. Support: FAPESP grants 2013/08135-2, 2019/18672-1, 2019/18702-8 and 2020/02043-2.