Therapeutic strategies based on CAR-T cells have revolutionized cancer immunotherapy, representing a promising option for several hematological malignancies. Nevertheless, despite the high remission rates after CAR-T cell administration, a significant number of patients still relapse, highlighting the need of improved therapeutic approaches. Of note, most CAR-T therapies tested nowadays rely on genetic T-cell engineering with integrating viral vectors that have several limitations that become, in some cases, insurmountable, such as the risks of malignant transformation and prohibitive costs. Moreover, autologous CAR-T therapies could also present limited efficacy, especially in heavily treated patients. Thus, our work is focused on the combination of state-of-the-art technologies to provide innovative and cost-effective CAR-T cell products.

The TranspoCART project has been conceived to develop a highly innovative CAR-T product by including novel modular multifunctional CAR designs, for enhanced safety and efficacy, as well as revolutionary virus-free gene-transfer strategies using Sleeping Beauty transposon systems. Altogether, these innovations have enabled the development of the TranpoCART19 cell product, a novel fully functional CAR-T cell approach targeting CD19, with increased safety and substantially reduced production costs, that is currently being evaluated in patients with non-Hodgkin lymphoma (NHL). Moreover, combining non-viral vectors and gene editing tools, we have developed HLA-I/TCR knockout CAR-T cells against CD33 for allogeneic CAR-T cell approaches in acute myeloid leukemia (AML). Our optimized protocol allows one-step generation of edited CAR-T cells showing similar phenotypic profiles than non-edited CAR-T cells, with equivalent in vitro and in vivo antitumoral efficacy. Transcriptional and genomics analyses revealed safe integration profiles of Sleeping Beauty transposons, with no preferences for specific genomic regions, and a highly specific editing of the HLA-I and TCR, with no off-targets.

In summary, our results demonstrate that the combination of CRISPR technologies with transposon-based vectors allow the generation of innovative and cost-effective CAR-T therapies compatible with clinical applications, representing a promising option for NHL and AML treatment.