Design and Testing of RNA Therapeutics to Block VEGFR2 and EGFR Expression in Human Glioblastoma

Abstract

Glioblastoma multiforme (GBM), is the most common and aggressive malignant primary brain tumor with a median survival of 15 months with standard care. Current therapies are limited by the blood brain barrier. Tumor blood vessel formation depends on vascular endothelial growth factor receptor 2 (VEGFR2), while tumor cell proliferation is stimulated by epidermal growth factor receptor (EGFR). Both are important for tumor cell survival. Our novel approach delivers directly to the tumor cells, genes encoding RNA therapeutics. Our strategy is to alter the splicing mechanism of the VEGFR2 and EGFR genes to block their activation, thus stop tumor cell angiogenesis and growth Our current RNA therapeutics demonstrate significant reductions in EGFR and VEGFR2 expression. To increase the efficacy of our RNA therapeutics, we are fine‐tune targeting regions of EGFR and VEGFR2, as well as recruiting splicing silencers and enhancers to the pre‐mRNA transcripts. Can we generate therapy vectors that encode antisense RNA therapeutics that either block or activate splicing motifs and alter the expression of functional VEGFR2 and EGFR in GBM significantly reducing tumor growth? Forty‐five antisense sequences were designed to target the EGFR gene and nine antisense sequences for VEGFR2, to potentially block their activation. The antisense sequences were cloned into our pAAV‐U7‐smOPT therapy vector. In addition, multiple strategies were used to clone the exonic splicing silencer 4G‐quadruplex and five distinct exonic splicing enhancer motifs into the RNA anti‐sense therapy vector, which recruit serine‐arginine (SR) proteins, directing the spliceosome to a desired splice site or intronic poly‐adenylation site. Moreover, another aspect of this research is to isolate the mRNA of multiple tyrosine kinase receptors from GBM cancer cells, clone the cDNA into a T7 expression vector to transcribe control RNA to use in our high throughput sequencing experiments. Multiple GBM cell lines, including U87 and SKMG3 were transfected with our novel therapies. Total mRNA was collected, analyzed, and compared to the same cell lines without treatment, showing a significant reduction in target expression. The collected data allow analysis to measure efficacy of current anti‐VEGFR2 and anti‐EGFR therapeutic strategies which will be tested in a mouse model using adeno‐associated virus (AAV) vector.