ABSTRACT: Allogeneic CD19-specific chimeric antigen receptor (CAR) T cells with inactivated donor T cell receptor (TCR) expression can be used as an 'off-the-shelf' therapeutic modality for lymphoid malignancies, thus offering an attractive alternative to autologous, patient-derived T cells. Current approaches for T cell engineering mainly rely on the use of viral vectors. Here, we optimized and validated a non-viral genetic modification platform based on Sleeping Beauty (SB) transposons delivered with minicircles to express CD19-28z.CAR and CRISPR/Cas9 ribonucleoparticles to inactivate allogeneic TCRs. Efficient TCR gene disruption was achieved with minimal cytotoxicity and with attainment of robust and stable CD19-28z.CAR expression. The CAR T cells were responsive to CD19+ tumor cells with antitumor activities that induced complete tumor remission in NALM6 tumor-bearing mice while significantly reducing TCR alloreactivity and GvHD development. Single CAR signaling induced the similar T cell signaling signatures in TCR-disrupted CAR T cells and control CAR T cells. In contrast, TCR disruption inhibited T cell signaling/protein phosphorylation compared to the control CAR T cells during dual CAR/TCR signaling. This non-viral SB transposon-CRISPR/Cas9 combination strategy serves as an alternative for generating next-generation CD19-specific CAR T while reducing GvHD risk and easing potential manufacturing constraints intrinsic to viral vectors.