Effects of antisense oligonucleotides targeting PD-1 pre-mRNA identified by DIRAC-dCas13-based screening on PD-1-positive lymphocytes

[Background and Purpose] In the tumor environment, T cells chronically express PD-1, and its interaction with PD-L1 on tumor cells results in decreased T cell cytokine secretion and proliferation. Exon2 of PD-1 encodes the PD-L1-binding domain. There have been few studies targeting RNA rather than the protein of PD-1 to upregulate lymphocyte antitumor immunity, especially the pre-mRNA region involved in Exon 2 splicing. We have recently reported that the region of PD-1 pre-mRNA was identified using the CRISPR/dCas13 system and a human CD8 + T cell line, and cytokine secretion capacity was maintained in the RNA region-targeted CD8 + T cells (PMID: 40920775). These findings were revealed in the presence of the idea that disrupting the interaction of the Exon2 splice cis-trans element on PD-1 pre-mRNA prevents the expression of the extracellular domain of PD-1, allowing lymphocytes to exert their inherent. In this presentation, we report on the effects on lymphocytes using a newly designed antisense oligonucleotides (ASO) derived from the identified pre-mRNA region of PD-1. [Materials and Methods] The designed PD-1 ASO sequences were based on the concept of the screening workflow of CLOVERNA Inc., and the ASO was provided by CLOVERNA Inc.. The ASO targeting PD-1 pre-mRNA was transduced into the human CD8 + T cell line, EBT-8 cells. The cells were maintained in GIT medium supplemented with recombinant human IL-2. Five days after transfection, the cells were harvested, and cell surface PD-1 expression was measured by flow cytometry. [Results] The new, unpublished data showed a decrease in the percentages of PD-1-positive cells of CD8 + T cells by the transfection with the ASO targeting PD-1 pre-mRNA. [Conclusion] This study revealed that the designed ASOs derived from the specific sequences of pre-mRNA identified using CRISPR/dCas13 contribute to suppressing PD-1 expression on the cell surface of human CD8 + T cells. [Future Plans] To assess tumor clearance capacity, we are in the preparation stage to begin in vitro cell-killing assays or in vivo PDX model experiments.