577. Distribution, Time Course, In-Vivo Imaging, Expression and Inflammatory Response after Non-Viral Gene Delivery to Rat and Primate CNS

Abstract

Top of pageAbstract Gene delivery may provide both pre-operative neuroprotection before procedures with a predictable high risk, as well as an effective therapy after post-traumatic or ischemic CNS or spinal cord injury. Both DNA and mRNA can be delivered by non-viral, cationic lipid-mediated methods 1, 2. We describe and quantitate extensive distribution, uptake, and cellular expression, including in vivo imaging, in the CNS of the rat of reporter genes such as GFP, luciferase, and Hsp70, in cells which include neurons. PCR and immunohistochemistry results confirm extensive distribution and uptake throughout primate CNS. Heat shock proteins (HSP) are rapidly induced by severe stress. The inducible Hsp70 is protective in whole animals, isolated organs and cells subjected to heat shock against a subsequent near lethal ischemic or hypoxic event. However, the induction of protective intracellular responses by heat shock is not clinically useful. Using methods identical to those for rodent experiments, a single injection of 50 |[mu]|g/kg of GFP-expressing DNA vector with cationic lipid (MLRI) was injected into free CSF in the cistern of anesthetized animals. After recovery from the anesthetic, normal behavior was observed for various times from 24 hours to 6 months, then sacrificed and perfused. At necropsy, blood, CSF and 50 tissues are analyzed for vector copies, and expression is characterized by immunohistochemistry of brain sections. Experiments in human CSF (2) confirm extended protection from degradation of mRNA by CNS RNases using these formulation techniques. In-vivo imaging in rat CNS (Xenogen IVIS) shows a peak at 72 hours after DNA delivery, with signal persisting to at least 18 days. mRNA expression in vivo has much more rapid onset, peak, and decay. Neuroprotection strategies designed for pre-operative use will also have significant application following stroke, brain or spinal cord trauma, or neurodegenerative disease. (See Table)