We have achieved widespread non-viral delivery, uptake, and expression in rat brain of Hsp70, the most highly inducible and neuroprotective of the Heat Shock Proteins, using novel lipid-based formulations of mRNA and DNA delivery vectors. We have recently confirmed widespread delivery by PCR of identical nonviral vectors throughout primate central nervous system (CNS). For further pre-clinical development, we must now test the efficacy of neuroprotection strategies, evaluate cellular toxicity including apoptosis, and quantify the expression of Hsp70 and other therapeutic transgenes in the CNS. Our ongoing program of mRNA and DNA vector development and CNS delivery in mammalian brain reflects established collaborations with experts in the various specialized fields that are critical for advances in this translational research, which by necessity encompasses molecular biology, lipid chemistry, animal models of neurophysiology, and finally, clinical medicine. To accomplish the objectives of this application we will subclone neuroprotective and anti-apoptotoic gene sequences into our established vectors and deliver these neuroprotective gene vectors in a well-established model for permanent focal ischemia. We will evaluate timing and dose response of mRNA and DNA, and quantify distribution and level of expression of the proteins using immunohisto- and fluorescence chemistry, double staining, stereology, and the laser scanning cytometer. Neuroprotection will be evaluated using measurement of infarct volumes, and neuroanatomic characterization will include measurement of apoptosis using TUNEL staining. If necessary, we will enhance targeting and control of therapeutic expression vectors by adding cell specific and regulatory elements into our existing, established, and highly successful Hsp70 and reporter gene sequences. We will develop biodegradable lipid delivery compounds with cellular or tissue targeting capabilities for reduced cytotoxicity. Neuroprotection strategies designed for pre-operative use will also have significant application for attenuation of secondary injury following stroke, brain or spinal cord trauma, or neurodegenerative diseases.
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