S346 Introduction. mRNA transfection and expression of reporter gene proteins in vivo in rat central nervous system (CNS) and in neuronal and non-neuronal cells using non-viral delivery have been described [1]. Cationic lipid/mRNA complexes stabilize the nucleic acid in the extracellular environment [2]. Stabilizing sequences in the optimized DNA construct, from which the mRNA is transcribed, serve the same function in the intracellular milieu [3]. These data support the feasibility of mRNA therapy for pre-operative expression of protective intracellular proteins. Heat shock proteins (HSP) are members of a highly conserved family of molecular chaperones, some of which are rapidly induced by heat stress. Pretreatment with heat shock leads to increased survival after ischemic and hypoxic stress in cells, perfused organs, and in whole animals [4]. Transient expression of the inducible, protective isoform of the HSP70, HSP70-1, also known as HSP72 [5], by delivery of mRNA is therefore hypothesized to be a means of improving cellular survival, and would be anovel application of gene transfer technology. Surgical patients present a unique situation, for whom a risk of surgical, ischemic, or hypoxic stress can be predicted. Despite the best anesthetic management, surgical manipulation during neurosurgical resection can create regional ischemia and hypoxic regions of brain. If hypotension and hyperventilation contribute to a decreased cerebral perfusion pressure, hypoperfusion can be even more significant, leading to areas of "watershed" neural tissue that is at risk for cell death [6]. The same can occur during cardiac bypass, due to an embolic shower of both gases and particulate from atherosclerotic vessel walls. Surgical procedures which require hypothermic circulatory arrest are also associated with neurological or neuropsychological deficits in the human. Risks of spinal cord ischemia leading to paraplegia after thoracic aneurysm surgery can be as high as 15%. Transient expression of protective intracellular proteins before a potentially severe stress could allow recovery of a greater fraction of the cells at risk. Methods. A variety of stabilized reporter mRNA transcripts encoded by HSP70-1, beta-galactosidase (beta-gal) and P. pyralis luciferase cDNA have been used to transfect neuronal and non-neuronal cells in culture. Transfections were optimized using novel cationic lipids [7] and formulation conditions [8]. Uptake and translation was demonstrated by chemiluminescent reaction assay of either beta-gal or luciferase and by Elisa and Western blot analysis of HSP70-1. Nucleic acid/cationic lipid complexes were then injected or infused into rat CNS parenchyma and ventricle and tissues were assayed for chemiluminescence. Results. Expression using mRNA in 30 to 45 minutes is seen, with a peak in expression at 6 hours. Levels of expression with mRNA transcripts compare well with DNA transfections, however DNA transfections take longer to reach similar levels of expression. Delivery, uptake and expression of HSP70-1 is demonstrated in vitro in neuronal cells and in vivo in rat brain. Formulation methods for in vivo transfection have been optimized [8] (see Table 1). Experiments which quantify protective effects of exogenous HSP70-1 expression are underway.Table 1: Results of in vivo rat brain transfections. Rats #1 and 2 were transfected with 2[micro sign]g DNA, all others received 2[micro sign]g mRNA. Injections were unilateral (#1-3) or bilateral (#4-6). Left ventricles were used as negative controls in #1-3. Luciferase in controls was not significantly above zero.Conclusions. Transfection with mRNA/cationic lipid complexes, which are self-limited, rapidly achieves significant expression of intracellular proteins. Results support the further development of mRNA-based HSP therapy for pre-operative protection from ischemic CNS damage.