Injection Of Lipoplexes Research Articles

Reducing off-target drug accumulation by exploiting a type-III interferon response.

Nanomedicines have been touted as the future of cancer therapy for decades. However, the field of tumor-targeted nanomedicine has failed to significantly advance toward becoming the primary choice for cancer intervention. One of the largest obstacles that has yet to be overcome is off-target accumulation of the nanoparticles. We propose a novel approach to tumor delivery by focusing on decreasing off-target accumulation of nanomedicines rather than directly increasing tumor delivery. Acknowledging a poorly understood "refractory" response to intravenously injected gene therapy vectors observed in ours and other studies, we hypothesize that virus-like particles (lipoplexes) can be utilized to initiate an anti-viral innate immune response that limits off-target accumulation of subsequently administered nanoparticles. Indeed, our results show a significant reduction in the deposition of both dextran and Doxil® in major organs with a concurrent increase in plasma and tumor accumulation when injection occurred 24h after a lipoplex injection. Furthermore, our data showing that the direct injection of interferon lambda (IFN-λ) is capable of eliciting this response demonstrates a central role for this type III interferon in limiting accumulation in non-tumor tissues.

Combined Inhibition of FOSL-1 and YAP Using siRNA-Lipoplexes Reduces the Growth of Pancreatic Tumor.

Simple SummaryIntercepting the molecular mechanisms implicated in pancreatic cancer progression can be an efficient therapeutic approach to treat this aggressive tumor. The Hippo pathway is a key mechanism driving tumor progression, even in the absence of KRAS activation. When this pathway is switched off, the transcriptional coactivator YAP is translocated into the nucleus and induces the activation of several genes implicated in tumor progression and apoptosis inhibition. FOSL-1 is a transcription factor that synergizes with YAP, forming a transcriptional complex. It has been shown to have a good therapeutic outcome when they are individually inhibited. In this work, we showed for the first time that the combined inhibition of YAP and FOSL-1 mRNA expression, using siRNA-lipoplexes, induces superior control over tumor growth in vitro and in vivo, compared to the individual treatments, and a reduction of the tumor stroma. The results offer a new therapeutic approach for pancreatic cancer treatment.Pancreatic cancer evades most of the current therapies and there is an urgent need for new treatments that could efficiently eliminate this aggressive tumor, such as the blocking of routes driving cell proliferation. In this work, we propose the use of small interfering RNA (siRNA) to inhibit the combined expression of FOSL-1 and YAP, two signaling proteins related with tumor cell proliferation and survival. To improve the efficacy of cell transfection, DODAB:MO (1:2) liposomes were used as siRNA nanocarriers, forming a complex denominated siRNA-lipoplexes. Liposomes and lipoplexes (carrying two siRNA for each targeted protein, or the combination of four siRNAs) were physico-chemically and biologically characterized. They showed very good biocompatibility and stability. The efficient targeting of FOSL-1 and YAP expression at both mRNA and protein levels was first proved in vitro using mouse pancreatic tumoral cell lines (KRASG12V and p53 knockout), followed by in vivo studies using subcutaneous allografts on mice. The peri-tumoral injection of lipoplexes lead to a significant decrease in the tumor growth in both Athymic Nude-Foxn1nu and C57BL/6 mice, mainly in those receiving the combination of four siRNAs, targeting both YAP and FOSL-1. These results open a new perspective to overcome the fast tumor progression in pancreatic cancer.

The Effect of Repeat Administration of Lipoplexes on Gene Delivery, Biodistribution, and Cytokine Response in Immunocompetent Tumor-Bearing Mice

It is becoming increasingly clear that the intravenous administration of nanoparticles elicits an immune response that compromises delivery efficiency and can be life threatening. This study investigated both the systemic and tissue-level cytokine response to repeat administration of lipoplexes coated with either lactose or PEG. We report that blood cytokine levels differ significantly from that observed in individual tissues. While we consistently observed a reduced cytokine response to lactosylated particles, this did not result in enhanced delivery or expression as compared to PEGylated formulations. We also document that repeat injection did not increase plasmid levels in the liver, lung, or spleen, but delivery to the tumor was enhanced under these conditions. In addition, we show that changes in neither blood nor tissue cytokines correlated strongly with reporter gene expression, and we observed relatively constant expression efficiencies (RLU/ng plasmid) across all tissues despite a considerably reduced cytokine response in the tumor. Together, these results indicate that both biodistribution and cytokine responses are dramatically altered by a repeat intravenous injection of lipoplexes, and that the mechanisms regulating reporter gene expression are not straightforward.

THU0081 MIR-17-5P REDUCES INFLAMMATION AND BONE EROSIONS IN COLLAGEN INDUCED ARTHRITIS MICE AND DIRECTLY TARGETS THE JAK-STAT PATHWAY IN RHEUMATOID ARTHRITIS FIBROBLAST-LIKE SYNOVIOCYTES.

Background:micro-RNAs (miR) are strong regulators of gene expression. Their involvement in RA key cytokines pathway regulation entitles them as important players in RA pathophysiology. The miR 17-92 cluster has been widely studied in cancer as they regulate cell apoptosis.Objectives:The aims of this study were to screen miR 17-92 cluster’s expression in different RA phenotypes (erosive and non erosive), further elucidate the mechanisms and direct targets involved in miR-17-5p anti-inflammatory role and to investigate miR-17-5p therapeutic effect in arthritis.Methods:A miR array was performed in synovial tissue from naïve erosive and non-erosive RA patients. Intra-articular delivery of miR-17 lipoplex was performed in collagen induced arthritis model in mice. Clinical, histological and structural effects were studied over the course of arthritis. In depth studies of miR-17 mechanisms of action were performed in primary RA-FLS isolated from RA synovial tissue.Results:Among others, miR-17-5p expression was reduced in erosive RA. miR-17 transfection in arthritic paws significantly reduced clinical inflammation. Moreover, synovial B cells, T cells, macrophages and polynuclear neutrophils infiltrates were significantly reduced. Structural damage was also decreased as shown by a reduction in the number of osteoclasts and erosion score by CT analysis. Pro-inflammatory cytokines of the IL-6 family, STAT3 target genes and IL-1β expression were also significantly reduced, but not TNF-alpha. miR17 directly targeted the 3’-untranslated region of STAT3 and JAK1. STAT3 and JAK1 mRNA and protein expression were reduced in RA-FLS following miR-17 transfection. STAT3 and JAK1 mRNA and activation of STAT3 as assessed by immunohistochemistry were also reduced in injected paws.Conclusion:We demonstrate an anti-inflammatory and anti-erosive role of miR-17 in vivo. This effect involves the suppression of the IL-6 family autocrine amplifying loop through the direct targeting of JAK1 and STAT3 as shown in RA-FLS.FigureEffect of miR-17 lipoplex injection in Collagen induced arthritis (CIA) mice’s ankles.Disclosure of Interests:None declared

Monitoring method for transgene expression in target tissue by blood sampling

In this study, we have developed a novel method to monitor transgene expression in tissues by blood sampling. We administered plasmid DNA (pDNA) encoding non-secretory form of firefly luciferase as a reporter gene and pDNA encoding secretable Gaussia princeps luciferase as a monitor gene simultaneously into mice. Good positive correlations were found between log-transgene expression of the reporter gene and the monitor gene in the treated muscle, between the monitor gene in the treated muscle and plasma, and consequently between the reporter gene in the treated muscle and the monitor gene in plasma after naked pDNA transfer into the muscle of mice. Such positive correlations were also found with gastric serosal surface instillation of naked pDNA, intravenous injection of lipoplex, and hydrodynamics-based injection of naked pDNA. We developed monitoring method of transgene expression in tissues by blood sampling, which was named ‘Therapeutic transgene monitoring (TTM)’, after ‘Therapeutic drug monitoring (TDM)’.

Efficient in vivo transfection and safety profile of a CpG-free and codon optimized luciferase plasmid using a cationic lipophosphoramidate in a multiple intravenous administration procedure

As any drug, the success of gene therapy is largely dependent on the vehicle that has to selectively and efficiently deliver therapeutic nucleic acids into targeted cells with minimal side-effects. In the case of chronic diseases that require a life-long treatment, non-viral gene delivery vehicles are less likely to induce an immune response, thereby allowing for repeated administration. Beyond the gene delivery efficiency of a given vector, the nature of nucleic acid constructs also has a central importance in gene therapy protocols.Herein, we investigated the impact of two firefly luciferase encoding plasmids on the transgene expression profile following systemic delivery of lipoplexes in mice, as well as their potential to be safely and efficiently readministered. Whereas pTG11033 plasmid is driven by a strong ubiquitous cytomegalovirus promoter, pGM144 plasmid, which has been designed to avoid inflammation and provide sustained transgene expression in lungs, is CpG-free and is under control of the human elongation factor-1 alpha promoter.Combined to the efficient cationic lipophosphoramidate BSV4, bioluminescence data showed that both plasmids were mostly expressed in the lungs of mice following a primary injection of lipoplexes. However, mice transfected with pGM144 exhibited a higher and more sustained transgene expression than those treated with pTG11033. Repeated administration studies revealed that several injections of lipoplexes could lead to similar transgene expression profiles if an interval of several weeks between subsequent injections was respected. A transient hepatotoxicity and a partial inflammatory response were caused by lipoplex injection, irrespective of the plasmid used.Altogether, these results indicate that repeated systemic administration of lipophosphoramidate-based lipoplexes in mice conducts to an effective lung transfection without serious side effects, and highlight the need to use long-lasting expressing and well tolerated plasmids in order to efficiently renew transgene expression by the successive doses.

The Co-Delivery of Oxaliplatin Abrogates the Immunogenic Response to PEGylated siRNA-Lipoplex

In vivo application of siRNA/PEGylated cationic liposome complex (lipoplex) is impeded by two main obstacles: cytokine responses and anti-PEG IgM responses to PEGylated siRNA-lipoplex. Here, we investigated whether co-administration of oxaliplatin (l-OHP) abrogates the cytokine release and anti-PEG IgM production by PEGylated siRNA-lipoplex. Free l-OHP was administered either simultaneously or 30 min prior to PEGylated siRNA-lipoplex administration, and cytokine response and anti-PEG IgM production were evaluated. In addition, the effect of the liposomal encapsulation of l-OHP on the immunogenic response of PEGylated siRNA-lipoplex was investigated. Simultaneous co-administration of free l-OHP with PEGylated siRNA-lipoplex caused a significant reduction in anti-PEG IgM production, along with an increase in the cytokine response. Free l-OHP injected prior to the lipoplex injection, however, successfully reduced cytokine release and anti-PEG IgM response. Platination of siRNA by simultaneously administered free l-OHP might facilitate the dissociation of double-stranded siRNA to single-stranded siRNA, resulting in the inducement of a potent immuno-stimulation of siRNA via endosomal toll-like receptors (TLRs). On the other hand, encapsulation of l-OHP into the siRNA-lipoplex resulted in a reduction of both anti-PEG IgM production and cytokine responses. Our results suggest that, besides the expected therapeutic efficacy of co-administration, encapsulation of l-OHP into the PEGylated siRNA-lipoplex has great potential for minimizing the immunostimulation of PEGylated siRNA-lipoplex, resulting in a safe, applicable, and compliant treatment regimen for sequential clinical administration.

The Role of Fibronectin in Pulmonary Gene Transfer Following Intravenous Administration of Lipoplex in Mice

We analyzed the effect of serum and fibronectin on pulmonary transgene expression after intravenous injection of cationic liposome-plasmid DNA (pDNA) complex (lipoplex) in mice. 1,2-Dioleoyl-3-trimethylammonium-propane (DOTAP) methyl sulfate salt/cholesterol lipoplex was incubated with several serum components for 5 min at 37°C prior to injection. We analyzed pulmonary transgene expression and pulmonary accumulation of lipoplex. While interaction with serum did not decrease pulmonary transgene expression, interaction with heat-inactivated serum did decrease it. Moreover, interaction with fibronectin enhanced pulmonary transgene expression. Inhibition of the binding of fibronectin to integrin decreased pulmonary transgene expression after injection of untreated lipoplex. We found that pulmonary accumulation of lipoplex changed depending on the kind of interacting serum components after injection. Furthermore, interaction with fibronectin increased pulmonary accumulation of lipoplex. Interaction with serum was required for pulmonary gene transfer following intravenous injection of lipoplex. Fibronectin appears to be a particularly critical component. Furthermore, the binding of fibronectin interacting with lipoplex to integrin was an important mechanism for pulmonary transgene expression.

Multiple components in serum contribute to hepatic transgene expression by lipoplex in mice

Interaction of cationic liposome/plasmid DNA complex (lipoplex) with serum was not a limiting factor for in vivo transfection. After intraportal injection of lipoplex, hepatic transgene expression was enhanced by interaction with serum in mice. In the present study, we analyzed the mechanism of enhanced hepatic transgene expression of lipoplex by interaction with serum components. Lipoplexes were incubated with several serum components for 5 min at 37 ° C before administration. Transfection efficiency of lipoplexes was measured 6 h after intraportal injection of lipoplex in mice. Depletion of divalent cation from serum decreased hepatic transgene expression. The addition of calcium ion to divalent cation-depleted serum restored transgene expression. Heat-inactivated serum and bovine serum albumin diminished the enhancing effect of serum on hepatic transgene expression. On the other hand, removal of anionic proteins from serum using an anion-exchanging column was critical for the enhancing effect of serum on transgene expression. Among the serum components tested, fibronectin and complement component C3 enhanced hepatic transgene expression. Hepatic transgene expression by lipoplex was enhanced by interaction with multiple components in serum. Interaction of lipoplex with serum could be an important factor for successful in vivo gene transfer. Hence, the information obtained in the present study is valuable for the future development of effective gene carriers.

Subconjunctival gene delivery of the transcription factor GA-binding protein delays corneal neovascularization in a mouse model

Corneal neovascularization can reduce visual acuity. GA-binding protein (GABP) is a transcription factor that regulates the expression of target genes including vascular endothelial growth factor (VEGF) and roundabout4 (Robo4), which participate in pathologic angiogenesis. We assessed whether intraocular injection of the GABP gene affects the growth of new corneal blood vessels in a mouse ocular neovascularization model. Transfection of human GABPalpha and GABPbeta gene (GABPalpha/beta) into human conjunctival epithelial cells resulted in decreased VEGF and Robo4 expression. Three groups of mice underwent chemical and mechanical denudation of the corneal epithelium. Subsequently, two groups were administered subconjunctival injection of lipoplexes carrying plasmid DNA encoding for human GABPalpha/beta or an empty plasmid DNA at 1-week intervals. The third group served as an experimental control. In vivo delivery of human GABPalpha/beta into mouse neovascularized cornea reduced VEGF and Robo4 gene expression. Biomicroscopic examination showed that, at 1 week after one or two injections, GABPalpha/beta-treated eyes had significantly less neovascularized corneal area than did eyes treated with the empty vector. Histologic examination showed significantly less vascularized area and fewer blood vessels in the GABP-treated group at 1 week after injections. However, these angiosuppressive effects were weakened at 2 weeks after injections. Our results indicate that subconjunctival GABP gene delivery delays corneal neovascularization for up to 2 weeks in a mouse model of deliberate corneal injury.

Cationic lipid/DNA complexes induce TNF-α secretion in splenic macrophages

Cationic lipids are widely used as vectors to deliver DNA into mammalian cells in vitro and in vivo. However, cationic lipid/DNA lipoplexes induce an inflammatory response, characterized by pro-inflammatory cytokine secretion, which severely limits their use. The main goal of this work is to identify the organs and the cell type involved in TNF-α secretion after lipoplex injection. We determined the kinetics of distribution of the cationic lipid/DNA complex in blood, lung, liver and spleen and quantified the TNF-α amount in organ homogenates and in the serum at different points of times. Increase in TNF-α production was only observed in the spleen and no significant increase of TNF-α production could be observed in the other organs. Fractionation of spleen cells revealed that macrophages were mainly responsible for TNF-α secretion. This observation was verified in vivo by using macrophage-removing agents. In conclusion, we show here that the TNF-α secreted in the serum after intravenous injection of lipoplexes comes mainly from the splenic macrophages.

Comparison of gene expression efficiency and innate immune response induced by Ad vector and lipoplex

Vectors for gene expression are the essential tools for both gene therapy and basic research. There are two groups of gene therapy vectors, viral and non-viral vectors. At present, toxicity triggered by vectors is one of the major concerns for clinical trials. In general, non-viral vectors, such as plasmid DNA–cationic liposome complex (lipoplex), are thought to be safer than viral vectors, such as adenovirus (Ad) vector, although lipoplex is less efficient in term of gene expression than the Ad vector. However, there has been no study directly comparing the gene expression efficiency and safety of viral and non-viral vectors. Here, we present evidence that the Ad vector shows much more efficient gene expression and is safer than lipoplex, at least with respect to the innate immune response. After being systemically administered to mice, the Ad vector showed a transduction efficiency that was 2 to 5 log orders higher than that of lipoplex, depending on the organ. On the other hand, surprisingly, the administration of lipoplex produced a greater amount of inflammatory cytokines such as interleukin-6, interleukin-12, and tumor necrosis factor-α than did the administration of the Ad vector, whereas a comparable level of hepatotoxicity was induced by these vectors. The production of inflammatory cytokines induced by the injection of lipoplex was reduced when the CpG motifs were removed completely from plasmid DNA. Thus, care should be taken to ensure the innate immune response induced by gene therapy vectors, especially lipoplex.

Mechanistic studies of sequential injection of cationic liposome and plasmid DNA

We previously reported that sequential injection of cationic liposome and plasmid DNA leads to notably reduced inflammatory toxicity and improved transfection in the lung (Y. Tan et al., 2001, Mol. Ther. 3, 673–682). The purpose of the current study was to explore the mechanism involved in sequential injection. It was observed that sequential injection resulted in dramatically lower DNA uptake by the liver and higher DNA levels in the lung than the lipoplex injection. In vitro experiments with macrophage cells further showed that sequential addition of liposomes and DNA could diminish the cellular uptake of DNA by these cells. The contributions of serum to the enhanced bioactivity and decreased toxicity were examined by injecting mice with samples of premixed liposome with serum and then DNA (LSD sample), and the resulting activities were compared to those obtained with injection of lipoplex–serum mixtures (LDS sample). LSD yielded 80% lower TNF-α levels and over 10-fold higher transfection than lipoplex, which is consistent with the reported findings with sequential injection. In contrast, LDS resulted in the same TNF-α levels and comparable transfection with lipoplex. Thus, the results suggest that the primary interaction of serum with liposome is a critical factor contributing to the superior activity and reduced toxicity of sequential injection. Studies on the interaction between mouse serum, liposomes, and DNA showed that DNA could bind negatively charged liposome–serum complex to form a ternary complex, which has a density similar to that of the ternary complex formed between lipoplex with serum. Further in vitro tests showed that LSD and LDS were similar in particle size and protein content, but different in protein composition as observed by 2-D gel electrophoresis. In addition, DNA in LSD was more readily displaced by dextran sulfate, an anionic polymer, than in LDS. The above findings suggest that the inhibition of opsonin protein binding on the particle surface with the sequential injection may contribute to the reduced macrophage uptake and cytokine induction and that the high ability of DNA release from the particles formed after sequential injection may contribute to the improved lung gene transfection.

1082. Delivery of Anti-Oxidative Enzyme Genes Protects Against Ischemia/Reperfusion-Induced Liver Injury in Mice

BACKGROUND: Hepatic ischemic/reperfusion (I/R) injury is characterized by the generation of reactive oxygen species (ROS), such as superoxide anions and hydrogen peroxide. The aim of this study is to investigate whether anti-oxidative gene delivery by our polycationic liposomes will be an effective approach for prevention of the injury. METHODS: Polycationic liposome-mediated extracellular superoxide dismutase (EC-SOD) or/and catalase gene delivery by portal vein injection was undertaken one day prior to warm I/R injury in mice. The effects of the gene delivery were determined 6 hours after starting reperfusion. RESULTS: Polycationic liposome-mediated anti-oxidative gene delivery led to marked hepatic transgene expression. There was a 56-fold increase in human EC-SOD gene expression in the liver 30 hours after injection of EC-SOD lipoplexes as shown by real time RT-PCR; Western blot analysis demonstrated a 10-fold increase in catalase protein levels in the liver as compared to controls. Liver SOD and catalase activity increased 16.6-fold and 9-fold, respectively. The overexpression of these two anti-oxidative genes significantly suppressed the subsequent I/R-induced acute liver injury as reflected by markedreduction of serum ALT levels (520±55 and 643±24 vs. 1217±193 units/ml, p<0.01). The I/R procedure significantly increased liver superoxide anions (699±83 vs. 20±13 RLU/sec/mg, p<0.01) as detected by a chemoluminescent method with a superoxide tracer, MCLA. These levels were markedly reduced to a nearly normal level (24±7 RLU/sec/mg) by the prior delivery of the EC-SOD gene. In addition, there were reduced liver malondialdehyde levels, and restored glutathione levels, in mice receiving either EC-SOD or catalase gene delivery. Moreover, the enhanced activation of nuclear factor-κB and AP-1 in hepatic I/R injury was minimized by the anti-oxidative gene delivery. The control lipoplexes did not affect the extent of liver injury and oxidant stress levels in this I/R model. The combination of EC-SOD and catalase lipoplex injection (in half amount of each plasmid) resulted in a slight improvement in hepatic I/R injury when compared to either alone. CONCLUSIONS: The findings demonstrate that polycationic liposome-mediated EC-SOD or catalase gene delivery led to high levels of the transgene activity in the liver, and markedly attenuated liver I/R injury. The protection is associated with enhanced anti-oxidative effects in the liver. To our knowledge, this is the first successful demonstration that polycationic liposome-mediated EC-SOD and/or catalase gene delivery ameliorates warm I/R-associated liver injury in mice. This approach may become a potential novel therapy to improve graft function and survival after liver transplantation.

Reversible Mode of Binding of Serum Proteins to DOTAP/Cholesterol Lipoplexes: A Possible Explanation for Intravenous Lipofection Efficiency

There are many indications that interaction of serum proteins with intravenously injected cationic lipoplexes disturbs lipofection in vitro and in vivo. However, transfection with certain lipid compositions such as N-[1- (2,3-dioleoyloxy)propyl]-N,N,N-trimethylammonium chloride (DOTAP)/cholesterol appears to be more resistant to serum and more efficacious. We investigated the mechanism of interaction between fluorescently labeled lipoplexes of the above composition and fluorescently labeled serum proteins. Fluorescence resonance energy transfer measurements in vitro indicate that serum proteins interact instantly and closely with the DOTAP/cholesterol lipoplexes. In accord with this, preinjection of fluorescently labeled serum into mice before injection of lipoplexes showed an immediate association of proteins with lipoplexes. Serum proteins colocalized with the lipoplexes in the lung vasculature; however, they dissociated from the cationic lipid as soon as 1 hr postinjection, probably because of displacement of serum proteins from lipoplexes by extracellular proteoglycans. Indeed, this displacement was imitated by heparin, a typical glycosaminoglycan, and could be explained by the inability of weakly acidic serum proteins to neutralize the DOTAP/cholesterol electrical surface potential psi0. The stability of the cationic lipid psi0 in serum could be a key reason for the high lung association and transfection efficiency with this formulation.

677. Polycationic Liposome-Mediated Extracellular Superoxide Dismutase Gene Delivery Leads to High Levels of the Transgene Expression and Prevents Acute Liver Injury in Mice

BACKGROUND: We have developed a formulation of polycationic liposomes that has been shown to be non-toxic, highly stable in the bloodsteam, and very effective in liver gene transfer in mice (Gene Therapy 2003; 10:180–187). Extracellular superoxide dismutase (EC-SOD) inhibits the generation of superoxide anions in the interstitial space of tissues, and may play a role in minimizing oxidative stress in liver injury. The aim of the present study is to investigate whether our polycationic liposome-mediated human EC-SOD gene delivery will protect against lipopolysaccharide (LPS)-induced liver toxicity in D-galactosamine (GalN)-sensitized mice. METHODS: Polycationic liposomes were generated from polycationic lipid (PCL) and cholesterol (Chol). Lipoplexes were formed by complexing liposomes with control plasmid (pEGFP-C1) or EC-SOD plasmid (pEGFP-C1-ECSOD) before use. Mice were injected with thyroid hormone (T3, 4 mg/kg, s.c.) two days before lipoplexes were injected via the portal vein. One day following lipoplex injection, mice were treated with GalN (500 mg/kg, i.p.) plus LPS (25 mg/kg, i.p.). Serum alanine aminotransferase (ALT), SOD activity, liver histology, glutathione (GSH) content and lipid peroxidation were evaluated one day after GalN/LPS exposure. Human EC-SOD gene expression in mouse liver tissue was determined by quantitative RT-PCR two days after lipoplex injection. RESULTS: somes and the control lipoplexes were stable in their size for three months at 4°C. Injection of T3, PCL-Chol liposomes or control lipoplexes did not cause a significant change in serum ALT levels. Real time quantitative RT-PCR analysis showed that human EC-SOD mRNA levels in mouse liver tissue in EC-SOD lipoplex-injected group were 55-fold higher than saline, liposome or control lipoplex controls when mouse -actin was employed as a house-keeping control gene. Serum ALT levels in animals receiving portal vein injections of EC-SOD lipoplexes plus GalN/LPS exposure were much lower than in those receiving normal saline, liposomes alone, or control lipoplexes plus GalN/LPS exposure (62583 vs. 150865, 153194, 1880520 units/ml, p<0.01). Liver histology showed much less hepatocyte death in EC-SOD lipoplex-treated group than liposome- or control lipoplex-treated groups. Serum SOD activity in animals receiving injection of EC-SOD lipoplexes were higher than in those treated with liposomes only, or control lipoplexes (28.8 3.3 vs. 15.2 0.9, 17.4 3.4 units/ml, p<0.05). Liver GSH content in animals receiving EC-SOD lipoplex injection was preserved with less lipid peroxidation, as indicated by a lower level of malondialdehyde content. CONCLUSIONS: liposome-mediated EC-SOD gene delivery leads to the marked transgene expression, and attenuates acute liver injury due to reduced oxidative stress in GalN/LPS-exposed mice. To our knowledge, this is a first report which demonstrates that polycationic liposome-mediated EC-SOD gene delivery to the liver represents a potential therapy for hepatotoxin-induced liver injury.

Therapeutic effect of intravenous delivery of lipoplexes containing the interferon-beta gene and poly I: poly C in a murine lung metastasis model.

We have evaluated and compared the efficacy of systemic administration of lipoplex formulations containing plasmids encoding IFN-beta or IFN-gamma, and a synthetic double-strand RNA poly I:poly C (pI:pC), a type I IFN inducer, in a lung metastasis model in which colon carcinoma CT-26 cells were inoculated intravenously into immunocompatible mice. Injection of lipoplexes containing plasmid DNA, regardless of IFN gene insertion, stimulated a transient increase in the serum concentration of proinflammatory cytokines such as tumor necrosis factor (TNF)-alpha and IFN-gamma, while injection of lipoplexes containing pI:pC led to a low level of TNF-alpha and undetectable IFN-gamma production. Furthermore, injection of these lipoplexes containing plasmids resulted in the production of a mixture of type I and type II IFNs, partly derived from the inserted IFN genes, in lung tissue cultures. In tumor-prophylactic experiments, intravenous injection of lipoplexes containing plasmid, regardless of IFN gene insertion, showed a significant reduction in lung metastatic nodules probably due to proinflammatory cytokines such as TNF-alpha and IFN-gamma nonspecifically induced by the CpG motifs in the plasmid and the type I IFNs produced. On the other hand, the antimetastatic effect of pI:pC-lipoplex seemed to be due mainly to IFN-beta induced by pI:pC. In established lung metastasis experiments, a single intravenous administration of lipoplexes containing IFN-beta gene or pI:pC, but not other lipoplexes, showed a significant therapeutic effect on the tumor metastasis: reduction in tumor nodules and prolongation of survival time of tumor-burden mice. The therapeutic effects were specifically impaired by anti-IFN-beta antibody treatment, indicating that IFN-beta produced by the lipoplexes played an important role in the suppression of established metastatic lung tumors. Thus, the local IFN-beta in the lung delivered by intravenous administration of lipoplex containing IFN-beta gene or pI:pC may be a convenient and useful method of inhibiting established metastatic lung tumors.

Expression of luciferase in selected organs following delivery of naked and formulated DNA to rainbow trout ( Oncorhynchus mykiss) by different routes of administration

In the present work, the expression of luciferase in selected organs following administration of DNA delivered as naked, liposome-formulated or chitosan-formulated by different routes of administration (intramuscular, intraperitoneal and intravenous injection, immersion and anal intubation) was studied in rainbow trout ( Oncorhynchus mykiss). The different formulations and routes of administration both influenced in which organs luciferase was expressed and the magnitude of expression. The highest expression levels of luciferase in the head kidney and liver were found after an intraperitoneal injection of lipoplex 2. In the spleen, the highest levels were detected after injection of naked DNA (intraperitonal or intramuscular) and lipoplex 2 (intraperitoneal). Following intravenous injection, naked DNA gave higher expression levels in the organs than the formulated plasmids and immersion and anal intubation were not effective routes of delivery as no expression of luciferase could be detected in any of the organs tested. Additionally, PCR using a primer specific for a 600 bp region of the luciferase gene pcDNA3-luc was used to assess the distribution of the plasmid itself after intramuscular and intraperitoneal injection. Positive amplification was obtained in spleen, head kidney, liver and muscle at the injection site following injection of formulated plasmids, while only muscle tissue from the injection site was positive when naked DNA was used.

The role of tissue macrophages in the induction of proinflammatory cytokine production following intravenous injection of lipoplexes.

Recent studies have demonstrated that intravenous administration of a plasmid DNA-cationic liposome complex (lipoplex) induced significant proinflammatory cytokine production in blood and inhibited transgene expression in pulmonary endothelial cells. In this study, we examined the effects of gadolinium chloride (GdCl(3)) pretreatment on the biodistribution and induction of proinflammatory cytokine production and transgene expression after intravenous injection of a lipoplex in mice. GdCl(3) is known to transiently deplete liver Kupffer cells and spleen macrophages after intravenous administration. Intravenous administration of a lipoplex triggers high levels of proinflammatory cytokine production, such as TNF-alpha, IFN-gamma and IL-12 in serum and a large amount of (32)P-labeled lipoplex accumulates in the liver 1 h after intravenous administration. However, pretreatment with GdCl(3) dramatically reduces serum levels of these cytokines and liver accumulation of the lipoplex. RT-PCR analysis showed that mRNA expression of TNF-alpha greatly increases in the liver and spleen after lipoplex injection and that pretreatment with GdCl(3) reduces mRNA expression in these organs. Messenger RNA expression of TNF-alpha in the liver occurs in non-parenchymal cells (sinusoidal endothelial cells and/or Kupffer cells). Inhibition of cytokine production by pretreatment with GdCl(3) leads to recovery of transgene expression in the lung following the second injection of lipoplex, which was reduced following the first injection of lipoplex. Thus, the present study demonstrates that tissue macrophages involving liver Kupffer cells and spleen macrophages are closely involved in TNF-alpha production following i.v. administration of the lipoplex. It is also suggested that avoiding lipoplex uptake and subsequent cytokine production by these cells would be a useful method of maintaining a high level of gene expression in the lung after repeated injections.

Liposome-mediated transfer of the bcl-2 gene results in neuroprotection after in vivo transient focal cerebral ischemia in an animal model.

Acute cerebral ischemia causes hypoxic neuronal cell death by necrosis and apoptosis. Expression of anti-apoptotic transgenes in ischemic brain may provide a useful therapeutic strategy for alleviation of postischemic damage. The present study investigates liposome-mediated transfer of the human bcl-2 protein in a rat model of focal transient ischemia due to middle cerebral artery (MCA) occlusion. Two different types of plasmid vectors were used for bcl-2 expression: one driven by the constitutive cytomegalovirus promoter (pCMV) and another based on the hypoxia-inducible human vascular endothelial growth factor promoter (pHRE). Cationic liposome/plasmid DNA complexes (lipoplexes) were injected directly into the cerebrospinal fluid (CSF) of rats immediately after MCA occlusion. The brains of treated and control animals were analyzed 48 h later. Infarct volumes and numbers of apoptotic cells were quantified. Occlusion of the MCA resulted in ipsilateral cerebral infarcts in all study animals. Transfer of the bcl-2 gene resulted in high level widespread protein expression in the case of the pCMV-bcl2 plasmid, while animals treated with the pHRE-bcl2 vector showed lower expression levels of bcl2 which were in addition limited to the ischemic area. Treatment with pCMV-bcl2, but not with pHRE-bcl2, was able to significantly reduce the infarct volume, which was 109 +/- 8 mm(3) for pCMV-bcl2, 152 +/- 29 mm(3) for pHRE-bcl2, and 155 +/- 18 mm(3) for control animals. Animals transfected with either vector showed a significant reduction in numbers of apoptotic cells in the infarct and penumbra area compared with controls. There were no short-term neurological side-effects of the CSF injection of lipoplexes or of bcl-2 expression. In conclusion, the hypoxia-inducible bcl-2 expression mediated by intrathecal lipoplexes may represent a novel, biologically safe and lesion-selective therapeutic approach for neuroprotection after acute cerebral ischemia. DOI: 10.1038/sj/gt/3301676