587. Genetic Delivery of a miRNA Cluster with Polycistronic siRNAs Reduces Expression of Epidermal Growth Receptor in Human Glioblastoma Cells

Abstract

Glioblastoma multiforme (GBM), the most common malignancy of the brain, is a grade IV astrocytoma. It is one of the most rapidly growing and invasive types of glial tumors. The standard therapy includes surgical removal, radiation and chemotherapy with a median survival of about 14 months. In addition, systemic therapies are limited by the blood-brain barrier. To bypass the barrier, we are developing a gene delivery strategy to inhibit the expression of tyrosine kinase receptors (TKR), which are commonly upregulated in GBM. One TKR, epidermal growth factor receptor (EGFR), is overexpressed in GBM leading to uncontrolled growth and proliferation. Our approach is to recruit the RNA interference pathway. Although small interfering RNAs (siRNAs) are often utilized to silence gene expression, exogenously expressed siRNAs are not an effective strategy to treat human disease due to both extracellular and intracellular nucleases as well as activation of cellular immunity against foreign nucleic acids. To bypass these degradatory mechanisms, we use a natural miRNA cluster genetic background to effectively deliver the DNA encoding multiple anti-EGFR siRNAs by inserting them into the tertiary structure of the miRNA cluster, miR-17-92. The anti-EGFR polycistronic miRNA cluster (pAAV-miR-IP1) expresses six siRNAs directed against EGFR, specifically targeting the extracellular ligand binding domain, transmembrane domain, intracellular tyrosine kinase domain and 3’ untranslated region of the EGFR transcript. The vector, pAAV-miR-IP1, was transfected into the human GBM cell lines, A172 and U87MG. Results demonstrate that pAAV-miR-IP1 was expressed at high levels with a subsequent reduction in EGFR mRNA expression. Additional strategies include using the polycistronic delivery mechanism to target multiple TKRs in addition to EGFR.