RNA Therapeutics for Brain Tumors: Targeting Pre‐mRNA Splicing Motifs to Generate Therapeutic Gene Isoforms

Abstract

Individuals diagnosed with glioblastoma multiforme (GBM) have a short life expectancy of 12‐15 months. This project is to develop therapies for effective and continuous drug delivery to the brain, targeting cancer‐driving genes. Tumor cell proliferation in GBM is often stimulated by epidermal growth factor receptor (EGFR) and is important for tumor cell survival. In our lab, we are developing RNA therapies to alter the splicing mechanism of EGFR to block its activation, thus stop tumor cell growth. Our approach uses an adeno‐associated virus gene transfer vector encoding RNA therapeutics targeting critical elements of the EGFR pre‐mRNA transcript. In this project, we cloned therapies into our therapeutic delivery platform and tested their efficacy to alter EGFR gene expression in tissue culture cells. We have found that our therapies have led to a shift in Intron 10A retention increasing alternative intronic polyadenylation generating a short soluble therapeutic decoy. Our antisense therapies targeted the EGFR pre‐mRNA transcript, specifically, regions of exon 10 and intron 10. Therapies were either designed to enhance recognition of the alternative intronic polyadenylation signal or block recognition of canonical exon 10/exon 11 splicing. We targeted the Exonic Splicing Enhancer (ESE) wild‐type 5’ Splice Site (wt‐5’SS) of exon 10, and the regions surrounding the alternative intronic polyadenylation signal. Each target showed a significant increase in intron retention. Targeting the ESE showed a 5‐fold increase in intron retention (p < 0.04), the wt‐5’SS was 15‐fold (p < 0.001), and the region surrounding the alternative intronic polyadenylation signal showed the strongest effect, with greater than 60‐fold increase in intron retention (p < 0.002). Currently, we are examining additional antisense therapies, as well as generation of the therapeutic soluble decoy protein using ELISA.