Cationic Liposome-mediated Gene Transfer Research Articles

The therapeutic influence of epsilon3 isoform of apolipoprotein E on pathophysiological mechanisms of secondary brain injury evolution after experimental traumatic brain injury

Purpose. To test the therapeutic potential of apolipoprotein E isoform epsilon3, whose synthesis in nerve tissue was induced by means of gene therapy, in an experimental traumatic brain injury (TBI) model, and to substantiate its mechanism of therapeutic action based on current literature data.Methods. To determine structural and functional changes associated with severe diffuse TBI, adult male Wistar rats were subjected to weight drop impact acceleration injury and evaluated at day 10 after trauma. A mixture of DOTAP liposomes and 25 µg of recombinant plasmid pCMV-APOE3 cDNA was infused intraventicularly immediately after TBI using ALZET osmotic pumps. The hippocampal CA1 regions were analyzed by means of light and electron microscopy, morphometry, immunocytochemical and confocal analysis using markers selective for neurons (NeuN) and astrocytes (GFAP). The neurofunctional outcome was assessed in an open field test.Results. The research data have shown that cationic liposome-mediated APOE3 gene transfer has a beneficial effect on hippocampal structure and ultrastructure, diminishes neuronal loss and severity of diffuse axonal injury, gliosis and microglial reaction, as well as aids regression of posttraumatic stress and anxiety.Conclusion. According to literature data, possible mechanisms of apoE therapeutic action are (1) normalization of neural cellular lipid component, (2) its role in regulating reactive gliosis and inflammatory response of CNS to injury, (3) antioxidant effects of apolipoprotein E, (4) regulation of cell death mechanisms, (5) influence of apolipoprotein E on differential gene expression and regulation of genomic response of CNS cells to injury

Local Transient Myocardial Liposomal Gene Transfer of Inducible Nitric Oxide Synthase Does Not Aggravate Myocardial Function and Fibrosis and Leads to Moderate Neovascularization in Chronic Myocardial Ischemia in Pigs

This study was designed to explore the effect of transient inducible nitric oxide synthase (iNOS) overexpression via cationic liposome-mediated gene transfer on cardiac function, fibrosis, and microvascular perfusion in a porcine model of chronic ischemia. Chronic myocardial ischemia was induced using a minimally invasive model in 23 landrace pigs. Upon demonstration of heart failure, 10 animals were treated with liposome-mediated iNOS-gene-transfer by local intramyocardial injection and 13 animals received a sham procedure to serve as control. The efficacy of this iNOS-gene-transfer was demonstrated for up to 7 days by reverse transcriptase-polymerase chain reaction in preliminary studies. Four weeks after iNOS transfer, magnetic resonance imaging showed no effect of iNOS overexpression on cardiac contractility at rest and during dobutamine stress (resting ejection fraction: control 27%, iNOS 26%; P = ns). Late enhancement, infarct size, and the amount of fibrosis were similar between groups. Although perfusion and perfusion reserve in response to adenosine and dobutamine were not significantly modified by iNOS-transfer, both vessel number and diameter were significantly increased in the ischemic area in the iNOS-treated group versus control (point score: control 15.3, iNOS 34.7; P < 0.05). Our findings demonstrate that transient iNOS overexpression does not aggravate cardiac dysfunction or postischemic fibrosis, while potentially contributing to neovascularization in the chronically ischemic heart.

Influence of liposomal transfection of apolipoprotein E3 gene on neurologic and cognitive deficits dynamics at experimental traumatic brain injury

The aim of study was to estimate gene therapy influence, that promotes synthesis induction of 3 apolipoprotein E (apoE3) in damaged brain tissue, on motor and memory dysfunction and learning disorders after experimental traumatic brain injury (TBI). Heavy TBI in rats was inflicted under overall anesthesia by free falling from1.5 mof load weighting450 g. The mixture of DOTAP liposome and 25 µg of plasmid vector pCMV•SPORT6 with cDNA of APOE3 gene was infused intraventricularly using ALZET osmotic pump. Posttraumatic neurologic deficits was estimated according to the scale of motor dysfunction at the first day of experiment before TBI infliction and from 1st to 7th day after trauma. Cognitive functions (spatial memory and learning) were tested in Morris water maze during 7–10 days after injury. The obtained results testified that cationic liposome-mediated APOE3 gene transfer caused quicker regress of neurologic and cognitive disorders after experimental TBI.

Antitumor activity of cationic liposome-mediated Bax gene transfer in osteosarcoma cells: Induction of apoptosis and caspase-independent cell death

The purpose of this study was to evaluate the anti-tumor effects of osteosarcoma (HOSM-1) cells via transfer of the Bax gene using a cationic liposome. We evaluated the levels of Bax, Bcl-xL, Bcl-2 and cytochrome c expression by Western blot analysis, and caspase-9 and -3 activities were determined in a colorimetric assay. Apoptosis was detected using a TUNEL assay, and cell growth inhibition was determined in an MTT assay. Following Bax gene transfer, release of cytochrome c to the cytosol was detected, the activities of caspase-9 and -3 increased, and TUNEL-positive cells (37.5%) were detected. Cell survival rate was 50.8% under these conditions. Induction of apoptosis was inhibited by a caspase inhibitor (zVAD-fmk), but only a slight increase in cell survival rate occurred. Hence, since not only apoptosis but also caspase-independent cell death is induced in HOSM-1 cells, we anticipate that Bax gene therapy with cationic liposomes will be useful for osteosarcoma.

Calorimetry of Cationic Liposome–DNA Complex and Intracellular Visualization of the Complexes

This chapter describes cationic liposome–mediated gene transfer that has become a widely used tool for in vitro transfection of eukaryotic cells and as a promising and safe alternative for in vivo gene therapy applications. It involves the formation of a cationic lipid–DNA complex that interacts efficiently with the cell surface, leading to the entry and expression of the exogenous DNA. The use of cationic lipids as DNA delivery systems for gene therapy is limited by the relatively low efficiency of the gene transfer process compared with viral vectors. Improvement of the in vitro and in vivo cationic lipid–mediated gene transfection efficiency can be attempted by use of trial and error approaches consisting of synthesizing and testing a large number of cationic lipid derivatives. Alternatively, a deeper understanding of the intermediate steps and mechanisms involved in the gene transfer process mediated by cationic lipids could allow the design of new rational strategies to improve the transfection efficiency. Two key steps are involved in the cationic lipid–mediated gene transfer process: (1) interaction between the cationic liposomes and DNA leading to the formation of a complex and (2) interaction between the complex and the target cells leading to DNA entry and expression of the transgene.

Lipoplex size determines lipofection efficiency with or without serum.

In order to identify factors affecting cationic liposome-mediated gene transfer, the relationships were examined among cationic liposome/DNA complex (lipoplex)-cell interactions, lipoplex size and lipoplex-mediated transfection (lipofection) efficiency. It was found that lipofection efficiency was determined mainly by lipoplex size, but not by the extent of lipoplex-cell interactions including binding, uptake or fusion. In addition, it was found that serum affected mainly lipoplex size, but not lipoplex-cell interactions, which effect was the major reason behind the inhibitory effect of serum on lipofection efficiency. It was concluded that, in the presence or absence of serum, lipoplex size is a major factor determining lipofection efficiency. Moreover, in the presence or absence of serum, lipoplex size was found to affect lipofection efficiency by controlling the size of the intracellular vesicles containing lipoplexes after internalization, but not by affecting lipoplex-cell interactions. In addition, large lipoplex particles showed, in general, higher lipofection efficiency than small particles. These results imply that, by controlling lipoplex size, an efficient lipid delivery system may be achieved for in vitro and in vivo gene therapy.

Folate-targeted, cationic liposome-mediated gene transfer into disseminated peritoneal tumors.

A folate-targeted, cationic lipid based transfection complex was developed and found to specifically transfect folate receptor-expressing cells and tumors. These liposomal vectors were comprised of protamine-condensed plasmid DNA, a mixture of cationic and neutral lipids, and a folic acid-cysteine-polyethyleneglycol-phosphatidylethanolamine (FA-Cys-PEG-PE) conjugate. Pre-optimization studies revealed that inclusion of low amounts (0.01 to 0.03%) of FA-Cys-PEG-PE yielded the highest binding activity of dioleoylphosphatidylcholine/cholesterol liposomes to folate receptor-bearing cells. In contrast, higher amounts (>0.5%) of FA-Cys-PEG-PE progressively decreased cellular binding of the liposomes. In vitro studies with cationic lipid/dioleoylphosphatidylethanolamine formulations indicated that as little as 0.01 to 0.3% of FA-Cys-PEG-PE was needed to produce optimal targeted expression of plasmid DNA. Similarly, using a disseminated intraperitoneal L1210A tumor model, maximum in vivo transfection activity occurred with intraperitoneally administered formulations that contained low amounts (0.01 mol%) of the FA-Cys-PEG-PE targeting lipid. Overall, folate-labeled formulations produced an eight- to 10-fold increase in tumor-associated luciferase expression, as compared with the corresponding non-targeted cationic lipid/DNA formulations. These results collectively indicate that transfection of widespread intraperitoneal cancers can be significantly enhanced using folate-targeted techniques.

Cationic liposome-mediated GDNF gene transfer after spinal cord injury.

Glial cell line-derived neurotrophic factor (GDNF) has been shown to protect cranial and spinal motoneurons, which suggests potential uses of GDNF in the treatment of spinal cord injury (SCI) and motor neuron disease. We examined neuroprotective effect of cationic liposome-mediated GDNF gene transfer in vivo on axonal regeneration and locomotor function recovery after SCI in adult rats. The mixture of DC-Chol liposomes and recombinant plasmid pEGFP-GDNF cDNA was injected after SCI. RT-PCR confirmed the increased expression of GDNF mRNA in the injected areas at 7 days after injection. The expression of EGFP-GDNF was observed in the cells around the injection locus by fluorescence microscope at least 4 weeks after injection. Four weeks after GDNF gene transfer, regeneration of the corticospinal tracts was assessed using anterograde tract tracing. There are more HRP labeling of corticospinal tract axons across the lesion in GDNF group compared with control group. In GDNF group, the maximum distance these labeled axons extended varied in different animals and ranged from 5 mm to approximately 9 mm from the lesion. In control group, no HRP labeled axons extended caudal to the lesion. The locomotion function of hindlimbs of rats was evaluated using inclined plane test and BBB locomotor scores. The locomotion functional scores in GDNF group were higher than that in control group within 1-4 weeks after SCI (p < 0.05). These data demonstrate that in vivo transfer of GDNF cDNA can promote axonal regeneration and enhance locomotion functional recovery, suggesting that cationic liposome-mediated delivery of GDNF cDNA may be a practical gene transfer method for traumatic SCI treatment.

Inhibition of nonviral cationic liposome-mediated gene transfer into primary human respiratory cells by interferon-gamma.

The effect of interferon (IFN) gamma on cationic liposome-mediated gene transfer into primary respiratory epithelial cells was investigated. Treatment of primary respiratory epithelial cells with IFN-gamma resulted in a dose-dependent increase in the intermediate filament cytokeratin 13 and a decrease in cellular proliferation, indicating that respiratory cells underwent squamous differentiation. IFN-gamma pretreatment resulted in a dramatic inhibition of transfection efficiency mediated by a cationic liposome (DOTAP). Incubation of squamous nasal cells with DOTAP/DNA complexes for various periods at 4 degrees C and evaluation of luciferase levels suggested that IFN-gamma pretreatment inhibits complex binding to the cells. In primary nasal and bronchial cells cytofluorimetric analysis demonstrated that IFN-gamma reduces binding of FITC-labeled complexes. The data indicate that differentiation of respiratory epithelial cells to a squamous phenotype, which may occur in chronic respiratory diseases such as cystic fibrosis, induces a refractory condition to gene transfer by nonviral cationic liposomes.

Evaluation of the role of lipoprotein metabolism genes in systemic cationic liposome-mediated gene transfer in vivo.

Germ line gene disruption and gene insertion are often used to study the function of selected genes in vivo. We used selected knockout and transgenic mouse models to attempt to identify lipoprotein-related genes and gene products that regulate the process of intravenous cationic liposome-DNA complex (CLDC)-based gene delivery. Several observations suggested that proteins involved in lipoprotein metabolism might be important in influencing the delivery and/or expression of CLDC. First, in vitro transfection of either K562 or CHO cells by CLDCs was enhanced by the presence of a functional low-density lipoprotein receptor (LDLR). Second, pretreatment of mice with 4-aminopyrazolopyrimidine (4APP), an agent that alters lipoprotein profiles in mice, significantly decreased expression of luciferase (luc) after intravenous injection of CLDC-luc complexes in mice. Therefore, we tested mouse model systems either deficient for, or overexpressing, selected genes involved in lipoprotein metabolism, for their potential to regulate intravenous, CLDC-based gene delivery. Although homozygous knockout mutation in the apoE gene caused a significant decrease in gene expression in many tissues of apoE-deficient mice, mice with homozygous deletion of both the apoE and LDLR genes showed wild-type levels of gene transfer efficiency. Thus, a secondary event, produced by homozygous deletion of apoE, but compensated for by the concomitant deletion of LDLR, and/or effects resulting from strain-related, genetic background differences, appeared to play a significant role in mediating intravenous, CLDC-based gene delivery. Secondary alterations resulting from germ line knockouts, as well as epigenetic effects produced by strain differences, may limit the ability to assign specific, gene transfer-related functions to the deleted gene.

Efficiency and Toxicity of Liposome-mediated Gene Transfer to Corneal Endothelial Cells

Gene transfer to corneal endothelial cells could be an important advance to modulate functions of these critical cells and is a field of current investigations. The development of gene transfer methods is a prerequisite for gene therapy to realize its full potential. We attempted to investigate and optimize the efficacy and safety of cationic liposome mediated gene transfer into corneal endothelial cells using different lipid formulations. Mono- and polycationic lipids and the neutral helper lipid dioleolphosphotidyl-ethanolamine (DOPE) were used for preparation of cationic liposomes. Six liposomal formulations containing DAC/DOPE 30/70 (DAC 30), DOSGA/DOPE 30/70 (DOSGA 30), DOSGA 100, DMRIE/DOPE 50/50 (DMRIE 50) and SP/DOPE 20/80 (SP 20) were complexed with the pUT 651-plasmid, encoding the E. coli beta-galactosidase gene. Subconfluent primary and passaged bovine corneal endothelial cells (BCEC) were transfected with different amounts of liposomes and DNA or uncomplexed free DNA as control. Quantitative expression of beta-galactosidase was measured using a colorimetric assay. In order to assess the effects on cell viability and growth, a modified acidic phosphatase assay was employed. Differences were detected using these various liposome preparations. Transfection experiments demonstrated the highest gene expression using SP 20> DMRIE 50 ranging at approximately 3mU per β-gal per well. Low expression of beta-galactosidase was achieved using DAC 30, DOSGA 30 and DOSGA 100. No beta-galactosidase expression was found in control dishes. There was no difference seen following transfection of primary or subsequent passages of BCEC. As indicated by the acid phosphatase assay, no significant toxicity was detected for the most efficient lipids used. Of the preparations studied, SP 20 appeared as the optimal vehicle for plasmid-mediated transfection of BCEC. The ability to deliver genes to BCEC via liposomes could be valuable, since the use of other vectors for transfection may be limited by undesired effects.

Tumor-targeted p53-gene therapy enhances the efficacy of conventional chemo/radiotherapy

A long-standing goal in gene therapy for cancer is a stable, low toxic, systemic gene delivery system that selectively targets tumor cells, including metastatic disease. Progress has been made toward developing non-viral, pharmaceutical formulations of genes for in vivo human therapy, particularly cationic liposome-mediated gene transfer systems. Ligand-directed tumor targeting of cationic liposome–DNA complexes (lipoplexes) is showing promise for targeted gene delivery and systemic gene therapy. Lipoplexes directed by ligands such as folate, transferrin or anti-transferrin receptor scFv, showed tumor-targeted gene delivery and expression in human breast, prostate, head and neck cancers. The two elements, ligand/receptor and liposome composition, work together to realize the goal of functional tumor targeting of gene therapeutics. The tumor suppressor gene, p53, has been shown to be involved in the control of DNA damage-induced apoptosis. Loss or malfunction of this p53-mediated apoptotic pathway has been proposed as one mechanism by which tumors become resistant to chemotherapy or radiation. The systemically delivered ligand–liposome–p53 gene therapeutics resulted in efficient expression of functional wild-type p53, sensitizing the tumors to chemotherapy and radiotherapy. This is a novel strategy combining current molecular medicine with conventional chemotherapy and radiotherapy for the treatment of cancer. The systemic delivery of normal tumor suppressor gene p53 by a non-viral, tumor-targeted delivery system as a new therapeutic intervention has the potential to critically impact the clinical management of cancer.

Efficacy of cationic liposome-mediated gene transfer to mesangial cells in vitro and in vivo.

Mesangial cells represent a major target for gene transfer approaches to the kidney. To establish a liposome-based system for transfection of mesangial cells we analyzed the efficacy and toxicity of different cationic liposomes and other nonviral transfection methods in primary cultures of rat and human mesangial cells using the Escherichia coli beta-galactosidase (lacZ) gene as a marker. In addition, an expression vector containing a human renin cDNA under the control of the cytomegalovirus immediate-early promoter/enhancer was generated, introduced into mesangial cells, and assayed in a system of transient gene expression. In vivo, gene transfer was studied after infusion of liposome/DNA complexes in the kidney of rats via the renal artery. Transfection efficiency ranged from 5.5% with DMRIE Liposomes in rat mesangial cells to 1.1% with LipofectAmine liposomes in human mesangial cells. Cytotoxicity following transfection was dependent on the transfection method. Transfection with the human renin expression vector led to the secretion of 11 pg/10(4) cells/48 h human renin in rat mesangial cells, 3,600 pg/10(4) cells/48 h in 293 cells, and 113 pg/10(4) cells/48 h human renin in opossum kidney cells. In vivo, infusion of liposomes was accompanied by nephrotoxicity and did not result in marker gene expression. Together the data demonstrate that cationic liposomes are useful tools for transferring genes into mesangial cells, including human mesangial cells. Cationic liposomes provide a functional system for the synthesis and secretion of human renin in mesangial cells and other mammalian kidney cells. The current limitation of the evaluated liposomes for an efficient in vivo gene transfer to mesangial cells is the toxicity upon intrarenal arterial administration.

Rat glioma cell death induced by cationic liposome-mediated transfer of the herpes simplex virus thymidine kinase gene followed by ganciclovir treatment.

We studied antitumor effects and cell death induced by cationic liposome-mediated gene transfer of the herpes simplex virus thymidine kinase (HSV-tk) gene followed by ganciclovir treatment in cultured rat T9 glioma cells and in experimental gliomas produced from this cell line. To transfer genes we used small unilamellar cationic liposomes containing N-(alpha-trimethylammonioacetyl)-didodecyl-D-glutamate chloride. Video-enhanced contrast differential interference contrast microscopy was used for morphologic observations of cultured cells. When we treated the cells or implanted gliomas with the liposomes and ganciclovir, a strong effect was seen against tumor cells, and survival of tumor-implanted rats was increased. Morphologically, cell death observed after HSV-tk gene/liposome and ganciclovir treatment in the cultured glioma cells included both apoptosis and necrosis. Introduction of the HSV-tk gene in a DNA-liposome complex followed by ganciclovir treatment induced both apoptosis and necrosis, which together resulted in a potent antitumor effect.

Cationic liposome-mediated gene transfer to rat salivary epithelial cells in vitro and in vivo.

Previously we have shown that gene transfer to salivary gland epithelial cells readily occurs via recombinant adenoviruses, although the response is short-lived and results in a potent host immune response. The aim of the present study was to assess the feasibility of using cationic liposomes to mediate gene transfer to rat salivary cells in vitro and in vivo. Initially, for transfection in vitro, we used two cationic liposome formulations (GAP-DLRIE/DOPE and DOSPA/DOPE) complexed with plasmid encoding human growth hormone (hGH) as a reporter gene. Thereafter, using GAP-DLRIE/DOPE, plasmids were transferred to rat salivary glands in vivo, and hGH levels measured in saliva, serum and gland extracts. Under optimal conditions, transfection of rat submandibular glands (SMGs) was consistently observed. Approximately 95% of the cells transfected with a plasmid encoding beta-galactosidase were acinar cells. Maximal hGH expression was obtained during the first 48 h post-transfection using a plasmid encoding the hGH cDNA and complexed with GAP-DLRIE/DOPE. hGH was detected in gland extracts and saliva, and occasionally in serum. No systemic or local gland pathology was consistently or significantly observed. The levels of the reporter gene product, hGH, obtained after GAP-DLRIE/DOPE-mediated gene transfer are considerably lower (<0.5%) than those achieved with adenoviral vectors (10(8) PFU). Nonetheless, cationic liposome-mediated gene transfer to salivary glands may be useful for potential therapeutic applications.

TGF-α antisense gene therapy inhibits head and neck squamous cell carcinoma growth in vivo

Unlike normal mucosal squamous epithelial cells, head and neck squamous cell carcinomas (HNSCCs) overexpress TGF-alpha mRNA and protein which is required to sustain the proliferation of HNSCC cells in vitro. To determine whether TGF-alpha expression contributes to tumor growth in vivo, cationic liposome-mediated gene transfer was used to deliver an antisense expression construct targeting the human TGF-alpha gene into human head and neck tumor cells, grown as subcutaneous xenografts in nude mice. The TGF-alpha antisense gene was immediately detected in the cytoplasm of the tumor cells, translocated to the nucleus by 12 h and remained localized to the nucleus for up to 3 days. Direct inoculation of the TGF-alpha antisense (but not the corresponding sense) construct into established HNSCC tumors resulted in inhibition of tumor growth. Sustained antitumor effects were observed for up to 1 year after the treatments were discontinued. Down-modulation of TGF-alpha was accompanied by increased apoptosis in vivo. These experiments indicate that interference with the TGF-alpha/EGFR autocrine signaling pathway may be an effective therapeutic strategy for cancers which overexpress this ligand/receptor pair.

Effect of DNA/liposome mixing ratio on the physicochemical characteristics, cellular uptake and intracellular trafficking of plasmid DNA/cationic liposome complexes and subsequent gene expression

In order to identify the important factors involved in cationic liposome-mediated gene transfer, in vitro transfection efficiencies by plasmid DNA complexed with DOTMA/DOPE liposomes at different DNA/liposome mixing ratios were evaluated using four types of cultured cells with respect to their physicochemical properties. Significant changes were observed in the particle size and zeta potential of the complexes as well as in their structures, assessed by atomic force microscopy, which depended on the mixing ratio. In transfection experiments, except for RAW 264.7 cells (mouse macrophages), efficient gene expression was obtained in MBT-2 cells (mouse bladder tumor), NLH3T3 cells (mouse fibroblasts) and HUVEC (human umbilical vein endothelial cells) at an optimal ratio of 1:5, 1:7.5 or 1:5, respectively. On the other hand, cellular uptake of the [ 32P]DNA/liposome complexes increased in all cell types with an increase in the mixing ratio, which was not reflected by the transfection efficiency. The cellular damage determined by MTT assay was minimal even at the highest DNA/liposome ratio (1:10), indicating that the lower gene expression level at the higher ratio was not due to cytotoxicity induced by the complex. An ethidium bromide intercalation assay showed that the release of plasmid DNA from the complex, following the addition of negatively charged liposomes, was restricted as the mixing ratio increased. Furthermore, confocal microscopic studies using HUVEC showed that the 1:5 complexes exhibited a dispersed distribution in the cytoplasm whereas a punctuate intracellular distribution was observed for the 1:10 complexes. This suggests that there was a significant difference in intracellular trafficking, probably release from the endosomes or lysosomes, of the plasmid DNA/cationic liposome complexes between these mixing ratios. Taken together, these findings suggest that the DNA/liposome mixing ratio significantly affects the intracellular trafficking of plasmid DNA complexed with the cationic liposomes, which is an important determinant of the optimal mixing ratio in cationic liposome-mediated transfection.

Highly efficient liposome-mediated gene transfer of inducible nitric oxide synthase in vivo and in vitro in vascular smooth muscle cells.

The efficient introduction of regulatory genes into vascular smooth muscle cells (SMCs) is one of the most promising options for gene therapy of cardiovascular diseases. Cationic liposome-mediated gene transfer may become a favorable transfection technique with regard to patient's safety for in vivo administration. However, this method until now has its limitation in a low transfection efficiency. Therefore, the present study was designed to improve cationic liposome-mediated transfection of rabbit vascular SMCs in vitro and in vivo, in order to enhance transfection efficiency and present an optimized system which may offer a potential therapeutic benefit for in vivo application. Optimized lipofection of rabbit SMCs with the mammalian expression vector pE-N1 and the reporter gene green fluorescent protein resulted in a mean transfection efficiency of about 50%. The unique transfection of rabbit SMCs in vitro and in vivo with the inducible isoform of human nitric oxide synthase (NOSII), using the same vector, resulted in a successful transient transcription and translation of a functionally active human NOSII in rabbit SMC, persisting 5-6 days. We could further demonstrate that the transfection procedure and the transgene product did neither induce necrosis nor apoptosis under the conditions chosen and did not result in the induction of endogenous NOSII of transfected SMCs. These findings indicate potential therapeutic relevance for this nonviral gene transfer system for in vivo gene therapy for cardiovascular diseases.

Effects of altering dosing on cationic liposome-mediated gene transfer to the respiratory epithelium.

Liposome-mediated gene transfer is currently sub-optimal with respect to both the extent and duration of transgene expression. We investigated whether simple changes in DNA dosing could enhance either of these outcomes. Increasing DNA doses produced highest transgene expression at an intermediate dose with toxicity observed at higher doses, thereby likely limiting expression. Adminis- tering an equivalent DNA dose in aliquots over a 1-3 day period resulted in significantly lower gene expression and did not increase the duration of expression. Administration at different times of the day (and hence wake/sleep cycles of the animals) did not alter gene expression. We conclude that such simple changes in dosing regimes are unlikely to contribute to improvements in gene transfer efficiency.

Liposome-mediated NGF gene transfection following neuronal injury: potential therapeutic applications.

We have systematically investigated the therapeutic potential of cationic liposome-mediated neurotrophic gene transfer for treatment of CNS injury. Following determination of optimal transfection conditions, we examined the effects of dimethylaminoethane-carbamoyl-cholesterol (DC-Chol) liposome-mediated NGF cDNA transfection in injured and uninjured primary septo-hippocampal cell cultures and rat brains. In in vitro studies, we detected an increase of NGF mRNA in cultures 1 day after transfection. Subsequent ELISA and PC12 cell biological assays confirmed that cultured cells secreted soluble active NGF into the media from day 2 after gene transfection. Further experiments showed that such NGF gene transfection reduced the loss of chol- ine acetyltransferase (ChAT) activity in cultures following calcium-dependent depolarization injury. In in vivo studies, following intraventricular injections of NGF cDNA complexed with DC-Chol liposomes, ELISA detected nine- to 12-fold increases of NGF in rat CSF. Further studies showed that liposome/NGF cDNA complexes could attenuate the loss of cholinergic neuronal immunostaining in the rat septum after traumatic brain injury (TBI). Since deficits in cholinergic neurotransmission are a major consequence of TBI, our studies demonstrate for the first time that DC-Chol liposome-mediated NGF gene transfection may have therapeutic potential for treatment of brain injury.