James G. Hecker

Stability of mRNA/Cationic Lipid Lipoplexes in Human and Rat Cerebrospinal Fluid: Methods and Evidence for Nonviral mRNA Gene Delivery to the Central Nervous System

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.

Lipid-mediated delivery of RNA is more efficient than delivery of DNA in non-dividing cells

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 vector-mediated 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. Lipid-mediated 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 3h 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 2-4 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 1h) of luciferase expression after transfection, a peak in expression 5-7h after transfection, and a return to baseline within 12h after transfection. After DNA delivery significant luciferase activity did not appear until 7h after transfection, but peak luciferase expression was always at least one order of magnitude higher than after RNA delivery. The peak expression after luciferase-expressing DNA delivery occurred 36-48 h 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 delivery results, which are shown as well. RNA delivery may therefore be more suitable for short-term transient gene expression due to rapid onset, shorter duration of expression and greater efficiency, particularly in non-dividing cells. Higher mean levels of expression per cell obtained following DNA delivery and the longer duration of expression confirm a continuing role for DNA gene delivery in clinical applications that require longer term transient gene expression.

The carboxyl-terminal domain of inducible Hsp70 protects from ischemic injury in vivo and in vitro

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 apoptosisinducing 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.

Antioxidant enzyme gene transfer for ischemic diseases

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.

Heat shock proteins as biomarkers for the rapid detection of brain and spinal cord ischemia: a review and comparison to other methods of detection in thoracic aneurysm repair

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.

The benefit of hydrophobic domain asymmetry on the efficacy of transfection as measured by in vivo imaging.

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.

Whole Animal In Vivo Imaging After Transient, Nonviral Gene delivery to the Rat Central Nervous System

We previously showed that a vector:lipid delivery system, comprised of a plasmid DNA vector and cationic lipid (lipoplex), when injected into the cerebrospinal fluid (CSF) of rats can deliver reporter genes in vivo efficiently and with widespread expression to the Central Nervous System (CNS). To further characterize this delivery system, we now present experiments that demonstrate the in vivo time-to-peak expression of the reporter gene, firefly luciferase. We infused a formulated lipoplex containing the lipid MLRI [dissymmetric myristoyl (14:0) and lauroyl (12:1) rosenthal inhibitor-substituted compound formed from the tetraalkylammonium glycerol-based DORI] and pNDluc, a luciferase vector, into CSF in the cisterna magna (CM) of the rat. Luciferase activity was followed over time by bioluminescence imaging after injection of luciferin. Our results show that luciferase activity in the CNS of rats is widespread, peaks 72 hours after injection into CM and can be detected in vivo for at least 7-10 days after peak expression. We further show that in contrast to injection into CSF, enzyme activity is not widely distributed after injection of the vector into brain parenchyma, emphasizing the importance of CSF delivery to achieve widespread vector distribution. Finally, we confirm the distribution of firefly luciferase in brain by immunohistochemical staining from an animal that was euthanized at the peak of enzyme expression.

Heat shock proteins HSP70 and HSP27 in the cerebral spinal fluid of patients undergoing thoracic aneurysm repair correlate with the probability of postoperative paralysis

An understanding of the time course and correlation with injury of heat shock proteins (HSPs) released during brain and/or spinal cord cellular stress (ischemia) is critical in understanding the role of the HSPs in cellular survival, and may provide a clinically useful biomarker of severe cellular stress. We have analyzed the levels of HSPs in the cerebrospinal fluid (CSF) from patients who are undergoing thoracic aneurysm repair. Blood and CSF samples were collected at regular intervals, and CSF was analyzed by enzyme-linked immunosorbent assay for HSP70 and HSP27. These results were correlated with intraoperative somatosensory-evoked potentials measurements and postoperative paralysis. We find that the levels of these proteins in many patients are elevated and that the degree of elevation correlates with the risk of permanent paralysis. We hypothesize that sequential measurement intraoperatively of the levels of the heat shock proteins HSP70 and HSP27 in the CSF can predict those patients who are at greatest risk for paralysis during thoracic aneurysm surgery and will allow us to develop means of preventing or attenuating this severe and often fatal complication.

A Flexible Method for the Conjugation of Aminooxy Ligands to Preformed Complexes of Nucleic Acids and Lipids

Attachment of targeted ligands to nonviral DNA or RNA delivery systems is a promising strategy that seeks to overcome the poor target selectivity generally observed in systemic delivery applications. Several methods have been developed for the conjugation of ligands to lipids or polymers, however, direct conjugation of ligands onto lipid– or polymer–nucleic acid complexes is not as straightforward. Here, we examine an oximation approach to directly label a lipoplex formulation. Specifically, we report the synthesis of a cationic diketo lipid DMDK, and its use as a convenient ligation tool for attachment of aminooxy‐functionalized reagents after its complexation with DNA. We demonstrate the feasibility of direct lipoplex labeling by attaching an aminooxy‐functionalized fluorescent probe onto pre‐formed plasmid DNA–DMDK lipoplexes (luciferase, GFP). The results reveal that DMDK protects DNA from degradation on exposure to either DNase or human cerebral spinal fluid, and that simple mixing of DMDK lipoplexes with the aminooxy probe labels the complexes without sacrificing transfection efficiency. The biocompatibility and selectivity of this method, as well as the ease of bioconjugation, make this labeling approach ideal for biological applications.

A Single Intrathecal Injection of DNA and an Asymmetric Cationic Lipid as Lipoplexes Ameliorates Experimental Autoimmune Encephalomyelitis

Intrathecal delivery of gene therapeutics is a route of administration that overcomes several of the limitations that plague current immunosuppressive treatments for autoimmune diseases of the central nervous system (CNS). Here we report intrathecal delivery of small amounts (3 μg) of plasmid DNA that codes for an immunomodulatory fusion protein, OX40-TRAIL, composed of OX40, a tumor necrosis factor receptor, and tumor necrosis factor related apoptosis inducing ligand (TRAIL). This DNA was delivered in a formulated nucleic acid-lipid complex (lipoplexes) with an asymmetric two-chain cationic lipid myristoyl (14:0) and lauroyl (12:1) rosenthal inhibitor-substituted compound (MLRI) formed from the tetraalkylammonium glycerol-based compound N-(1-(2,3-dioleoyloxy)-propyl-N-1-(2-hydroxy)ethyl)-N,N-dimethyl ammonium iodide. Delivery and expression in the CNS of OX40-TRAIL in the mouse prior to onset of experimental autoimmune encephalomyelitis (EAE), an animal model for multiple sclerosis, decreased the severity of clinical disease. We believe this preclinical demonstration of rapid, widespread, and biologically therapeutic nonviral gene delivery to the CNS is important in further development of clinical lipid-based therapeutics for CNS disorders.