Gene Therapy for Brain Tumors: Identification of New Therapeutic Targets Based on RNA Structure

Abstract

This project is to develop therapies to bypass challenges to effective and continuous drug delivery to the brain, for the treatment of glioblastoma multiforme (GBM). Currently, individuals diagnosed with GBM have a short life expectancy of 12‐14 months. Our approach has the potential to deliver one single dose of gene therapy directly to the GBM tumor environment and block the production of 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. We have examined the ‘pre‐mRNA structurome’ of EGFR to evaluate the accessibility of targetable regions. To advance our therapeutic strategy, we have analyzed the secondary structure of the EGFR transcript 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 a cryptic polyadenylation signal. Based on these structural profiles, we generated RNA therapies that interact with structural elements to unravel the hidden polyadenylation signal with the potential to activate expression of the short therapeutic isoform. In this project, we cloned these therapies into our therapeutic delivery platform and tested their efficacy to alter EGFR gene expression in tissue culture cells. Currently, we are evaluating in vitro, the therapeutic RNA interaction with the target sequence of the EGFR pre‐mRNA transcript.