Poster abstracts
Poster number 108 submitted by John Kipp
Regulation of pd-l1 expression through splice-switching oligonucleotide treatment.
John Kipp (Center for Life Science EducationCenter for Childhood Cancer, Abigail Wexner Research Institute at Nationwide Childrens Hospital), Akila S. Venkataramany (Medical Scientist Training Program and Biomedical Sciences Graduate Program, OSUCOM; Center for Childhood Cancer, Abigail Wexner Research Institute at Nationwide Childrens Hospital), Kevin Cassady, MD (Dept. of Pediatrics, Div. of Pediatric Infectious Diseases, Nationwide Childrens Hospital; Center for Childhood Cancer, Abigail Wexner Research Institute at Nationwide Childrens Hospital), Dawn S. Chandler, PhD (Center for Childhood Cancer, The Research Institute at Nationwide Childrens Hospital; Dept. of Pediatrics, OSUCOM; Molecular, Cellular and Developmental Biology Graduate Program, OSU)
Abstract:
Programmed death-ligand (PD-L1), a transmembrane protein that interacts with the PD-1 receptor, is highly expressed in various pediatric malignancies and serves as a mechanism for tumor cells to evade immune response and develop resistance against endogenous apoptotic pathways and current immunotherapies. Anti-PD-L1 monoclonal antibody therapies have been clinically successful, yet limited response in other cancers and debilitating side effects in patients highlight a need for novel PD-L1-based therapies. Targetable regions include the IgV-like extracellular domain in exon 2, which is responsible for correct cellular localization and interaction with the PD-1 receptor. We hypothesize that down-regulation of PD-L1 expression by forcing defective pre-mRNA splicing of exon 2 will be a viable approach to ablate PD-L1 immunosuppressive activity in the tumor.
To test our hypothesis, we designed splice-switching oligonucleotides (SSOs) that block the 5’ and 3’ splice sites of PD-L1 exon 2. Skipping PD-L1 exon 2 would promote the expression of a non-functional PD-L1 pre-mRNA transcript and down-regulate the PD-L1 protein. We transfected Rh30 cells, which detectably express PD-L1, with either 5’, 3’, or 5’+3’ SSOs for 24 hours and harvested RNA to measure endogenous PD-L1 RNA levels and exon 2 skipping via RT-PCR. Simultaneously, we designed and successfully cloned a chimeric PD-L1 minigene, which includes exon 2 and the flanking intronic regions. We will transfect this minigene in HeLa cells to further validate our SSOs and study the regulation of PD-L1 alternative splicing.
While this project is in its beginning stages, the results from our study will be the foundation for developing an SSO therapy targeting PD-L1 expression in pediatric cancers. In addition, understanding the utility of RNA-based therapies that manipulate alternative splicing will be beneficial for novel therapies for any cancer with PD-L1-induced immunotherapy resistance.
Keywords: PD-L1, splice-switching oligonucleotides