Poster abstracts

Poster number 79 submitted by John Kipp

Regulation of PD-L1 expression through antisense oligonucleotide treatment.

John Kipp (Center for Childhood Cancer, The Research Institute at Nationwide Childrens Hospital), Akila S. Venkataramany (The Ohio State University College of Medicine Medical Scientist Training Program), Dr. Dawn Chandler (Center for Childhood Cancer, The Research Institute at Nationwide Childrens Hospital, Department of Pediatrics, The Ohio State University College of Medicine)

Abstract:
Programmed death-ligand (PD-L1) is a gene often shown to be highly expressed in various malignancies and has been associated with the programmed cell death of host immune cells. Though the currently approved anti-PD-L1 monoclonal antibody therapies are successful in select cancers, such as MPNST, novel PD-L1-based therapies are still needed to expand treatment options for other cancers and limit side effects for patients. Expression of PD-L1 leads to a cell membrane bound ligand that renders T-cells inactive through a binding interaction with the T-cell PD-1 receptor. PD-L1 splice variants with exclusion of exon 2 alter the ligand’s ability reach the cell membrane, therefore, preventing its normal function. We hypothesize that down-regulation of PD-L1 protein expression by forcing defective pre-mRNA splicing at the translational start site (exon 2) will be a viable approach to ablate PD-L1 immunosuppressive activity in the tumor
We are testing our hypothesis by using splice-switching oligonucleotides (SSOs) that block the 5’ and 3’ splice sites of PD-L1 exon 2. We transfected either 5’, 3’, or 5’+3’ SSOs in Rh30 cells, which express low-moderate levels of PD-L1, for 24 hours and observed a minimal splicing switch. Now, we are moving to more clinically relevant MPNST cell lines (5NPCIS and CT2A) that have higher PD-L1 expression. We have also designed a PD-L1 mini-gene that includes the first three exons of PD-L1 and intronic flanking regions using InFusion cloning to study PD-L1's splicing regulation before validating our SSOs and the splicing biology in an endogenous settingThis line of research may be able to identify an effective splice-switching strategy in PD-L1 that could benefit future treatment options. These results can be significant in clinical treatments as gene splicing therapies can coincide with existing therapies, while also serving as an alternative treatment option for cancers that have developed resistance to PD-L1 immunotherapies.

References:
Montes, M., Sanford, B. L., Comiskey, D. F., & Chandler, D. S. (2019). RNA Splicing and Disease: Animal Models to Therapies. Trends in genetics : TIG, 35(1), 68–87. https://doi.org/10.1016/j.tig.2018.10.002
Fabrizio, F. P., Trombetta, D., Rossi, A., Sparaneo, A., Castellana, S., & Muscarella, L. A. (2018). Gene code CD274/PD-L1: from molecular basis toward cancer immunotherapy. Therapeutic advances in medical oncology, 10, 1758835918815598. https://doi.org/10.1177/1758835918815598
Atsushi Kosaki, James Nelson, Nicholas J.G. Webster, Identification of Intron and Exon Sequences Involved in Alternative Splicing of Insulin Receptor Pre-mRNA*, Journal of Biological Chemistry, Volume 273, Issue 17,1998, Pages 10331-10337, ISSN 0021-9258, https://doi.org/10.1074/jbc.273.17.10331

Keywords: Splice-switching oligonucleotides, PD-L1, Alternative Splicing