584. Gene Delivery to Primary Neuronal Cells

Abstract

Top of pageAbstract Neuronal protection under numerous clinical circumstances (i.e. trauma brain injury, stroke, ischemia and circulation arrested surgery, etc.) remains a tough challenge due to the paucity of effective clinical options. Transient gene delivery to CNS neuronal cells may offer an alternative approach to induce neuronal protection. We have previously shown widespread distribution, uptake, and expression in rat brain after non-viral, cationic lipid-mediated delivery of reporter gene vectors to the lateral ventricle 1,2. In order to further develop techniques for, and understand the mechanisms of, non-viral gene delivery, we conducted cationic lipid mediated gene delivery of fire fly luciferase mRNA and DNA to primary rat cortical cells to study the post-transfection transgene expression profiles. The delivery of mRNA resulted in a rapid onset of luciferase expression 1 hour after transfection, peaked at 5- 6 hours post transfection, and diminished to base line 12 hours after transfection. Compared to the delivery of mRNA, DNA delivery resulted in a much later onset of gene expression. Although the transgene expression after DNA delivery was generally one order of magnitude higher than after mRNA delivery, the earliest significant luciferase activity after DNA transfection did not appear until 7 hours after transfection. Peak expression following DNA delivery was 36-48 hours after transfection, and transgene expression persisted at a significant level for at least one week before it dropped to base line. Based on this observation, we attempted the delivery of mRNA and DNA vectors that encode for the heat shock protein gene HSP70. The inducible HSP70 is a chaperone protein and its expression has protective effects under stress conditions. HSP70 was delivered using non-viral cationic lipid-mediated techniques that we have developed to rat primary cortical cells, and analyzed by flow cytometry and immunohistochemistry. Consistent with the luciferase activity results, we detected evenly stained HSP70 expression 5 hours after the delivery of HSP70 mRNA, while expression after the delivery of HSP70 DNA appeared to peak at 36-48 hours.