Production of Universal CAR-T cells against the αβ-TCR with All-in-one Nanoblades

Nanoblades are virus-like particles that contain a gRNA and a Cas9 protein to target a region of interest in the genome. In my project, we are making new All-in-one Nanoblades that contain a CAR RNA that is knocked into a targeted region. Our All-in-one Nanoblades are used to knock out the endogenous αβ TCR of T cells and simultaneously knock in a CAR that targets the αβ TCR. We have already produced All-in-one Nanoblades and demonstrated good efficiency of the CAR. After transducing primary T cells, we plan to sequence the integration site to verify the correct integration of our CAR DNA and to exclude the possibility of off-target sites. At the same time, we will use flow cytometry to check the presence of the endogenous αβ TCR and our CAR on the cell surface. After improving our transduction, we will analyse the surface markers of our CAR T cells to see if knocking out the αβ TCR has an effect on other proteins expressed in the T cells. Finally, we will test the functionality of these CAR-T cells using cytotoxicity and degranulation assays. The CAR-T cells we have generated could be used as Universal CAR-T cells, as they should have low fratricide and no GvH-reaction.

Publication History

Article published online:
09 May 2025