Splicing manipulation as a new strategy to enhance adoptive cell therapy
2Sharett Institute of Oncology, Hadassah Medical Hospital, Israel
3Department of Biochemistry and Molecular Biology, Hebrew University of Jerusalem, Israel
Alternative splicing of immune-receptors is a post-transcriptional process that can generate isoforms with a changed modulatory effect compared to the full-length receptor. To exemplify the impact of alternative splicing on the function of T-cells, we chose the PD-1 receptor. PD-1 has to main isoforms: full-length PD-1 (flPD-1) and soluble PD-1 (sPD-1). We postulated whether these variants could have opposing effects since flPD-1 is a well-documented inhibitor of T-cell activation, whereas the soluble form can block PD-L1, PD-1’s ligand.
To elucidate the role of the splicing isoforms of PD-1, we developed a system that interferes with splicing by targeting selected splice sites in T-cells. Using the CRISPR/Cas9 editing system, we introduced a double-strand break into a pre-defined splicing recognition sequence of the PD-1 gene. This manipulation increased exon 3 skipping and lowered the transcript level of flPD-1, although it did not abolish the gene entirely. As a result, sPD-1 was highly expressed in the T-cell supernatant.
Remarkably, when activated, the modified cells had significantly improved cytokine secretion and killing capcity. In contrast, knocking down PD-1 did not lead to an enhanced phenotype, suggesting that the improvement was indeed due to the soluble PD-1 and not the loss of the PD-1 receptor.
In summary, our research shows that the preferential expression of sPD-1 was essential for the improved T-cell function. As PD-1 plays a critical role in the failure of cancer immunotherapies, the potential of splicing manipulation of PD-1 can be valuable and applicable for the success of advanced cell-based therapies.