The design of appropriate gene delivery systems is essential for the successful application of gene therapy to clinical medicine. Cationic lipid-mediated delivery is a viable alternative to viral vectormediated gene delivery in applications where transient gene expression is desirable. However, cationic lipid-mediated delivery of DNA to post-mitotic cells such as neurons is often reported to be of low efficiency, due to the presumed inability of the DNA to translocate to the nucleus. Lipidmediated delivery of RNA is an attractive alternative to non-viral DNA delivery in some clinical applications, because transit across the nuclear membrane is not necessary. Here we report a comparative investigation of cationic lipid-mediated delivery of RNA versus DNA vectors encoding the reporter gene green fluorescent protein (GFP) in Chinese Hamster Ovary (CHO) and NIH3T3 cells following chemical inhibition of proliferation, and in primary mixed neuronal cell cultures. Using optimized formulations and transfection procedures, we assess gene expression by flow cytometry to specifically address some of the advantages and disadvantages of lipid-mediated RNA and DNA gene transfer. Despite inhibition of cell proliferation, over 45% of CHO cells express GFP after lipid-mediated transfection with RNA vectors. Transfection efficiency of DNA encoding GFP in proliferation-inhibited CHO cells was less than 5%. Detectable expression after RNA transfection occurs at least 3 hours earlier than after DNA transfection, but DNA transfection eventually produces a mean level of per cell GFP expression (as assayed by flow cytometry) that is higher than after RNA transfection. Transfection of proliferation-inhibited NIH3T3 cells and primary mixed neuronal cultures produced similar results, with RNA encoded GFP expression in 2 to 4 times the number of cells as after DNA encoded GFP expression. These results demonstrate the increased efficiency of RNA transfection relative to DNA transfection in non-dividing cells. We used firefly luciferase encoded by RNA and DNA vectors to investigate the time course of gene expression after delivery of RNA or DNA to primary neuronal cortical cells. Delivery of mRNA resulted in rapid onset (within 1 hour) of luciferase expression after transfection, a peak in expression 5-7 hours after transfection, and a return to baseline within 12 hours after transfection. After DNA delivery significant luciferase activity did not appear until 7 hours after transfection, but peak luciferase expression was always at least one order of magnitude higher than after RNA delivery. The peak expression after luciferaseexpressing DNA delivery occurred 36-48 hours after transfection and remained at a significant level for at least one week before dropping to baseline. This observation is consistent with our in-vivo Publisher's Disclaimer: This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will u...

Heat shock protein (Hsp)70 can suppress both necrosis and apoptosis induced by various injuries in vivo and in vitro. However, the relative importance of different functions and binding partners of Hsp70 in ischemic protection is unknown. To explore this question, we tested the ability of Hsp70-K71E, an adenosine triphosphate (ATP)ase-deficient point mutant, and Hsp70-381-640, a deletion mutant lacking the ATPase domain and encoding the carboxyl-terminal portion, to protect against ischemia-like injury in vivo and in vitro. Heat shock protein 70-wild type (-WT), -K71E, -381-640, and control vector plasmid LXSN were expressed in primary murine astrocyte cultures. Astrocytes overexpressing Hsp70-WT, -K71E, or -381-640 were all significantly protected from 4 h combined oxygen-glucose deprivation and 24 h reperfusion when assessed by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay or propidium iodide staining and cell counting (P < 0.05). Brains of rats were transfected with plasmids encoding Hsp70-WT, -K71E, -381-640, or LXSN 24 h before 2 h middle cerebral artery occlusion followed by 24 h reperfusion. Animals that overexpressed either of the mutant proteins or Hsp70-WT had significantly better neurological scores and smaller infarcts than control animals. Protection by both mutants was associated with reduced protein aggregation, as assessed by ubiquitin immunohistochemistry and reduced nuclear translocation of apoptosis-inducing factor. The results show that the carboxyl-terminal portion of Hsp70 is sufficient for neuroprotection. This indicates that neither the ability to fold denatured proteins nor interactions with cochaperones or other proteins that bind the amino-terminal half of Hsp70 are essential to ischemic protection.

The heat shock proteins (HSPs) are members of highly conserved families of molecular chaperones that have multiple roles in vivo. We discuss the HSPs in general, and Hsp70 and Hsp27 in particular, and their rapid induction by severe stress in the context of tissue and organ expression in physiology and disease. We describe the current state of knowledge of the relationship and interactions between extra-and intracellular HSPs and describe mechanisms and significance of extracellular expression of HSPs. We focus on the role of the heat shock proteins as biomarkers of central nervous system (CNS) ischemia and other severe stressors and discuss recent and novel technologies for rapid measurement of proteins in vivo and ex vivo. The HSPs are compared to other proposed small molecule biomarkers for detection of CNS injury and to other methods of detecting brain and spinal cord ischemia in real time. While other biomarkers may be of use in prognosis and in design of appropriate therapies, none appears to be as rapid as the HSPs; therefore, no other measurement appears to be of use in the immediate detection of ongoing severe ischemia with the intention to immediately intervene to reduce the severity or risk of permanent damage.

Clinical applications of gene therapy require advances in gene delivery systems. Although numerous clinical trials are already underway, the ultimate success of gene therapies will depend on gene transfer vectors that facilitate the expression of a specific gene at therapeutic levels in the desired cell populations without eliciting cytotoxicity. In clinical applications for which transient expression is desirable, mRNA delivery is of particular interest. We have shown cationic lipid-mediated mRNA delivery to be feasible, efficient, and reproducible in vitro. mRNA delivery to the cerebrospinal fluid (CSF) in vivo would provide a means of vector distribution throughout the central nervous system (CNS). This study examined the functional integrity and protection from degradation of mRNA/cationic complexes (lipoplexes) in human cerebrospinal fluid (hCSF) in vitro and expression of these lipoplexes in vivo. Results obtained from gel electrophoresis indicate that cationic lipids protect mRNA transcripts from RNases in hCSF for at least 4 hr. This is in contrast to the total disappearance of nonlipid-complexed mRNA in less than 5 min. We confirmed the importance of RNase activity by incubating mRNA transcripts encoding luciferase or green fluorescent protein (GFP) in hCSF to which RNase inhibitors had been added. After incubation, these solutions were used to transfect Chinese hamster ovary (CHO) cells in vitro. Next, assays for both GFP and luciferase were used to demonstrate functional integrity and translation of the mRNA transcripts. Finally, we delivered in vitro transcribed mRNA vectors encoding for Hsp70 and luciferase to the lateral ventricle of the rat in a series of preliminary in vivo experiments. Initial immunohistochemistry analysis demonstrates that the distribution, uptake, and expression of reporter sequences using lipid-mediated mRNA vector delivery is extensive, as we earlier reported using similar methods with DNA vectors but that the expression may be less intense. Expression was noted in coronal sections throughout the rat brain, confirming the potential for lipid-mediated mRNA delivery to the CNS. These findings confirm that complexing mRNA with cationic lipid before exposure to CSF confers protection against RNase activity, facilitating distribution, cellular uptake, and expression of mRNA delivered into the CNS.

The balance of redox is pivotal for normal function and integrity of tissues. Ischemic insults occur as results of a variety of conditions, leading to an accumulation of reactive oxygen species (ROS) and an imbalanced redox status in the tissues. The oxidant stress may activate signaling mechanisms provoking more toxic events, and eventually cause tissue damage. Therefore, treatments with antioxidants, free radical scavengers and their mimetics, as well as gene transfer approaches to overexpress antioxidant genes represent potential therapeutic options to correct the redox imbalance. Among them, antioxidant gene transfer may enhance the production of antioxidant scavengers, and has been employed to experimentally prevent or treat ischemic injury in cardiovascular, pulmonary, hepatic, intestinal, central nervous or other systems in animal models. With improvements in vector systems and delivery approaches, innovative antioxidant gene therapy has conferred better outcomes for myocardial infarction, reduced restenosis after coronary angioplasty, improved the quality and function of liver grafts, as well as outcome of intestinal and cerebral ischemic attacks. However, it is crucial to be mindful that like other therapeutic armentarium, the efficacy of antioxidant gene transfer requires extensive preclinical investigation before it can be used in patients, and that it may have unanticipated short- or long-term adverse effects. Thus, it is critical to balance between the therapeutic benefits and potential risks, to develop disease-specific antioxidant gene transfer strategies, to deliver the therapy with an optimal time window and in a safe manner. This review attempts to provide the rationale, the most effective approaches and the potential hurdles of available antioxidant gene transfer approaches for ischemic injury in various organs, as well as the possible directions of future preclinical and clinical investigations of this highly promising therapeutic modality.

We, and others, have observed that the structure of cationic lipids appears to have a significant effect on the transfection efficacy of optimized nucleic acid/cationic lipid complexes (lipoplexes) used for in vitro and in vivo gene delivery and expression. Although there are many in vitro comparisons of lipid reagents for gene delivery, few comparisons have been made in vivo. We previously reported the effects of changes in hydrophobic domain chain length and chain asymmetry, changes in headgroup composition, and counterion exchange. We have observed in our own work over many years the apparent superiority of asymmetric versus symmetric hydrocarbon domains for otherwise similar lipids. In this investigation we use in vivo whole animal brain imaging to evaluate the contribution of symmetric versus asymmetric hydrophobic domains on what we previously determined to be optimal chain lengths for in vitro transfections. We specifically investigated several glycerol-based lipids; however, the rare reports of asymmetric non-glycerol-based lipids also support our observations. We found that asymmetric, two-chain cationic lipids of 14 to 18 carbons perform significantly better in vivo, as analyzed by whole animal imaging, than the paired symmetric lipids.

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The use of DNA for nonviral gene expression depends on several factors. These include (i) delivery and accessibility to the targeted tissue, (ii) protection from extracellular degradation, (iii) sufficient uptake by cells of interest, and (iv) protection from intracellular degradation to allow translation of adequate levels of intracellular or secreted proteins. As an initial step in demonstrating the feasibility of nonviral, cationic lipid-mediated gene therapy, we present evidence for the successful delivery and expression of heat shock protein Hsp70 and reporter gene enzymes in the central nervous system (CNS) of the rat after injection into the lateral ventricle. Gene delivery is accomplished using optimized formulations of plasmid DNA, which have been complexed with the cationic lipid MLRI. Results from DNA vectors encoding for green fluorescent protein (GFP), luciferase, and Hsp70 are reported. Standard immunofluorescent methods were used to demonstrate widespread expression of the reporter proteins and of Hsp70. Stereology analysis has been completed on three coronal sections, which illustrates the distribution of expression along the longitudinal axis. These initial findings support the further development of nonviral, lipid-mediated gene delivery technology for transient expression of protective, intracellular proteins and represent an important step leading to in vivo studies to identify potential clinical benefits.