| dc.description.abstract | Chimeric antigen receptor (CAR) T-cell therapy is a cancer therapy in which a patient’s own T-cells are engineered to express the chimeric antigen receptor, with which they can recognise and kill cancer cells. The therapy is extremely successful in B-cell cancers, but has some disadvantages as well. Currently, CAR T-cells are produced using viral vectors. Viral vectors integrate the CAR construct in the host DNA, resulting in permanent CAR expression, which can contribute to side-effects like B-cell aplasia, cytokine release syndrome, and insertional mutagenesis. In addition to side-effects, viral vectors are expensive and have a long production process, raising both the costs and time of CAR T-cell therapy. As an alternative, CAR mRNA can be used for safer, cheaper and faster CAR T-cell generation. To avoid the use of viral vectors, non-viral delivery strategies, such as lipid nanoparticles, can be explored to deliver the RNA or other cargo molecules into the T-cells. Since T-cells are resistant to chemical transfection, research is needed to improve transfection of T-cells with LNPs. CD44, a receptor involved in adhesion and migration, is expressed on both inactive and active T-cells and could be targeted to improve transfection. The anionic polymer hyaluronic acid (HA) is the main ligand of CD44. Here, we explore electrostatic coating of HA on cationic LNPs to improve the delivery of mRNA to primary T-cells. After HA coating, the transfection of primary T-cells by the cationic LNPs decreased instead of increasing. Parameters such as interference from PEGylated lipids, HA-CD44 binding on T-cells, and stability of HA coating were tested to rule out their role in the decrease of transfection efficiency. Coating with an alternative anionic polymer without CD44-affinity showed that the charge switch from cationic to anionic is most likely responsible for the decrease in transfection. HA coating on neutral LNPs did not decrease transfection, but neither did it increase. It is hypothesised that an excess of free HA present in the solution after coating is blocking the HA coated LNPs from binding to CD44. To conclude, we did not improve transfection of primary T-cells. However, we provided valuable information into factors influencing the effect of HA coating. In the future, this information can help to develop successful HA-coated LNPs for T-cell transfection and contribute to the development of safer and more accessible CAR T-cell therapy. | |
