Radiation Enhances the Delivery of Antisense Morphalino Oligonucleotides across the Blood-Brain Barrier

Abstract

We previously demonstrated that non-ablative ionizing radiation enhanced the delivery of intravenously administered unmodified antisense morpholino oligonucleotides (aONs) to subcutaneous tumor sites and results in 50% reduction in the DNA repair enzyme O6-methylguanine DNA methyltransferase (MGMT), compared to non-radiated subcutaneous tumor sites in the same animal. Delivery of aONs across the blood-brain barrier (BBB) remains a major challenge. Ionizing radiation is well known to alter BBB permeability. We hypothesized that ionizing radiation will facilitate the delivery of IV aONs across the BBB. Long Evans rats received either 5 Gy whole brain radiation, or no radiation (n=6 per group); 24 h later, half of each group received GFP-tagged nonspecific unmodified morpholino aON (10.5 mg/kg IV). Brains were harvested after perfusion with saline 24h after aON administration. GFP localization was assessed in 50 μm sections using a laser-scanning confocal microscope. We observed increased delivery of the morpholino aONs into the brain parenchyma only in the pre-radiated cohort. Rats that received radiation followed by IV GFP-conjugated aONs showed a punctate green fluorescent signal intracellularly in perivascular cells. The punctate staining pattern detected is suggestive that radiation enhanced the endocytosis of aONs and uptake into endosomes and lysosomes. Radiation enhances the targeted delivery of unmodified aONs across the BBB and uptake by perivascular cells. This observation is logical as radiation is known to enhance cellular endocytosis. Morpholino aONs are stable at physiologic pH and can be tailored to silence many key cancer proteins. This approach holds significant translational potential since radiation is already an integral part of brain tumor therapies. Follow-up studies will investigate if radiation enhances the targeted delivery of aONs in a rodent MGMT expressing-brain tumor model to inhibit MGMT expression and function as a chemo-radiation sensitizer.