Chimeric antigen receptor (CAR) T cell therapy is a revolutionary strategy to treat tumors that relies on genetic modification of autologous T cells to target a specific tumor antigen. Although this promising therapy has demonstrated clinical effectiveness against some hematologic malignancies, its autologous nature limits its universal use. To create a universal medicinal CAR-T cell product two main problems should be addressed: graft versus host disease and host versus graft response.

In this study allogeneic CAR-T cells were generated through β-2-microglobulin (B2M) gene disruption, a key component of the class I HLA (HLA-I) complex which allows the exposure of HLA-I on the cell surface. B2M gene disruption was performed using cytosine base editors (BE), a modified Cas9 that produces a single strand break directed by a single guide RNA (sgRNA), enabling the introduction of point mutations, minimizing indel generation. In addition, CAR construct was introduced using the Sleeping beauty (SB) transposon system.

BE in combination with a B2M sgRNA showed an efficacy of 95.2% in disrupting the B2M by flow cytometry which has been also confirmed by Sanger sequencing. Moreover, CAR introduction through the SB system demonstrated an efficacy of 58.4%. Altogether, allogeneic CAR-T cells generation exhibited an efficacy of 47.1%. Genetic modifications introduced to generate allogeneic CAR-T cells did not alter the proportion of T cell subpopulations and these allogenic CAR-T cells demonstrated in vitro functionality against tumor cell lines. This approximation reveals the feasibility of producing functional allogenic CAR-T cells using a combination of BE and transposons.