EXTH-26. SECONDARY STRUCTURE ANALYSIS OF THE EGFR PRE-mRNA TRANSCRIPT TO IDENTIFY TARGETABLE REGIONS AND OPTIMIZE RNA THERAPY

Abstract

Abstract Individuals diagnosed with glioblastoma multiforme (GBM) have a short life expectancy of 12–15 months. Current strategies are often limited by the blood-brain barrier. This project is to develop therapies to bypass challenges to effective and continuous drug delivery to the brain, targeting cancer-driving genes. Epidermal growth factor receptor (EGFR) is dysregulated in 57% of all GBM. Our approach uses an adeno-associated virus gene transfer vector encoding RNA therapeutics targeting critical elements of the EGFR pre-mRNA transcript. The ‘pre-mRNA structurome’ can be used to uncover and determine the accessibility of targetable regions. Our approach has the potential to deliver one single dose of gene therapy directly to the GBM tumor environment and block the production of EGFR and activate the expression of a stable therapeutic isoform of EGFR. To advance our therapeutic strategy, we have analyzed the EGFR secondary structure using selective 2’ hydroxyl acylation and primer extension followed by mutational profiling (SHAPE-MaP). SHAPE-MaP reactivity profiles were generated revealing the structure of splicing and cryptic polyadenylation signal (PAS) elements within the targeted region. We identified enhancer binding motifs surrounding the 5’ splice site and hidden elements of the cryptic PAS. Based on these structural profiles, we generated RNA therapies to unravel the hidden PAS to activate expression of the short therapeutic isoform.