ABSTRACTWidespread treatment of human diseases with gene therapies necessitates the development of gene transfer vectors that integrate genetic information effectively, safely and economically. Accordingly, significant efforts have been devoted to engineer novel tools that i) achieve high-level stable gene transfer at low toxicity to the host cell; ii) induce low levels of genotoxicity and possess a ‘safe’ integration profile with a high proportion of integrations into safe genomic locations; and iii) are associated with acceptable cost per treatment and scalable/exportable vector production to serve large numbers of patients. The Sleeping Beauty (SB) transposon has been transformed into a vector system that is fulfilling these requirements.In the CARAMBA project, we use SB transposition to genetically modify T cells with a chimeric antigen receptor (CAR) specific for the SLAMF7 antigen, that is uniformly and highly expressed on malignant plasma cells in multiple myeloma. We have demonstrated that SLAMF7 CAR-T cells confer specific and very potent anti-myeloma reactivity in pre-clinical models, and are therefore preparing a Phase I/IIa clinical trial of adoptive immunotherapy with autologous, patient-derived SLAMF7-CAR T cells in multiple myeloma (EudraCT Nr. 2019-001264-30/CARAMBA-1).Here we report on the characterization of genomic safety attributes in SLAMF7 CAR-T cells that we prepared in three clinical-grade manufacturing campaigns under good manufacturing practice (GMP), using T cells that we obtained from three healthy donor volunteers. In the SLAMF7 CAR-T cell product, we determined the average transposon copy number, the genomic insertion profile, and presence of residual SB100X transposase. The data show that the SLAMF7 CAR transposon had been inserted into the T cell genome with the close-to-random distribution pattern that is typical for SB, and with an average transposon copy number ranging between 6 and 12 per T cell. No residual SB100X transposase could be detected by Western blotting in the infusion products. With these attributes, the SLAMF7 CAR-T products satisfy criteria set forth by competent regulatory authorities in order to justify administration of SLAMF7 CAR-T cells to humans in the context of a clinical trial. These data set the stage for the CARAMBA clinical trial, that will be the first in the European Union to use virus-free SB transposition for CAR-T engineering.DisclosuresThis project is receiving funding from the European Union’s Horizon 2020 research and innovation programme under grant agreement No 754658 (CARAMBA).
Unexpectedly High Copy Number of Random Integration but Low Frequency of Persistent Expression of the Sleeping Beauty Transposase After Trans Delivery in Primary Human T Cells