Gene editing technologies for the improvement of the production of CD19-CAR-T cell-derived exosomes 

B Perucha(1) P Heredia(1) K Pavlovic(1,2) M Cortijo(1) V Ronco(1) F Martin(1) I C Herrera(2,3) J A Marchal(4,5,6) M D Carmona(2) K Benabdellah(1)

1:GENyO- Centro de Genomica e Investigacion Oncologica: Pfizer / Universidad de Granada / Junta de Andalucia; 2:GC14 Cell Therapy, IMIBIC. University of Cordoba, Reina Sofia University Hospital; 3:Department of Hematology, Reina Sofía University Hospital, Córdoba, Spain. Biomedical Research Institute (IBS. Granada); 4:Biopathology and Regenerative Medicine Institute (IBIMER), Centre for Biomedical Research (CIBM); 5:Department of Human Anatomy and Embryology, Faculty of Medicine, University of Granada; 6:Biosanitary Research Institute of Granada (ibs.GRANADA), University Hospitals of Granada - University of Granada

Chimeric antigen receptor (CAR) expressing T cells (CAR-T cells) have showed a great potential for the treatment of leukemias and lymphomas. However, CAR-T based therapies have also reported some limited efficacy attributed to insufficient CAR-T cells penetrance and/or persistence and due to the immunosuppressive tumor microenvironment as well. As a complementary approach, preclinical research has demonstrated that nanotechnology based in secreted exosomes can overcome some of these challenges. CAR-T cell-derived exosomes have been proposed as an emerging and complementary approach, that retain most of the CAR-T cells features, including the capacity of release of high number of cytotoxic molecules, low immunogenicity and high biocompatibility. However, several limitations must be acknowledged and addressed before they can be effectively used in a clinical application. One of the main limitations in the development of exosome-based therapies is the difficulty in producing enough and efficient amounts of therapeutic exosomes. With the aim to solve this last limitation we developed an effective and safe platform, based on genomic editing techniques, to enhance exosomes production from CAR-T cells. Our study provided a proof of concept with preliminary results that demonstrate the utility of our approach. We demonstrated that the knockout of gene 1 induces an increase in the amount of exosome production, according to transmission electron microscopy (TEM), nanoparticle tracking analysis (NTA) and atomic force microscopy (AFM). We also showed that the obtained exosomes carry CAR molecules on their surface. All our data, open the door to combinatorial immunotherapy approaches pairing  CAR-T with therapeutical EXO-CART.