Mechanism of plasmid delivery by hydrodynamic tail vein injection. I. Hepatocyte uptake of various molecules

BACKGROUND: The hydrodynamic tail vein (HTV) injection of naked plasmid DNA is a simple yet effective in vivo gene delivery method into hepatocytes. It is increasingly being used as a research tool to elucidate mechanisms of gene expression and the role of genes and their cognate proteins in the pathogenesis of disease in animal models. A greater understanding of its mechanism will aid these efforts and has relevance to macromolecular and nucleic acid delivery in general. METHODS: In an attempt to explore how naked DNA enters hepatocytes the fate of a variety of molecules and particles was followed over a 24-h time frame using fluorescence microscopy. The uptake of some of these compounds was correlated with marker gene expression from a co-injected plasmid DNA. In addition, the uptake of the injected compounds was correlated with the histologic appearance of hepatocytes. RESULTS: Out of the large number of nucleic acids, peptides, proteins, inert polymers and small molecules that we tested, most were efficiently delivered into hepatocytes independently of their size and charge. Even T7 phage and highly charged DNA/protein complexes of 60-100 nm in size were able to enter the cytoplasm. In animals co-injected with an enhanced yellow fluorescent protein (EYFP) expression vector and fluorescently labeled immunoglobulin (IgG), hepatocytes flooded with large amounts of IgG appeared permanently damaged and did not express EYFP-Nuc. Hepatocytes expressing EYFP had only slight IgG uptake. In contrast, when an EYFP expression vector was co-injected with a fluorescently labeled 200-bp linear DNA fragment, both were mostly (in 91% of the observed cells) co-localized to the same hepatocytes 24 h later. CONCLUSIONS: The appearance of permanently damaged cells with increased uptake of some molecules such as endogenous IgG raised the possibility that a molecule could be present in a hepatocyte but its transport would not be indicative of the transport process that can lead to foreign gene expression. The HTV procedure enables the uptake of a variety of molecules (as previous studies also found), but the uptake process for some of these molecules may be associated with a more disruptive process to the hepatocytes that is not compatible with successful gene delivery.     

Hydrodynamics-based procedure involves transient hyperpermeability in the hepatic cellular membrane: implication of a nonspecific process in efficient intracellular gene delivery

BACKGROUND: The mechanisms underlying the efficient gene transfer by a large-volume and high-speed intravenous injection of naked plasmid DNA (pDNA), a so-called hydrodynamics-based procedure, remain unclear and require further investigation. In this report, we have investigated possible mechanisms for the intracellular transport of naked pDNA by this procedure. METHODS: Propidium iodide (PI), a fluorescent indicator for cell membrane integrity, and luciferase- or green fluorescent protein (GFP)-expressing pDNA were injected into mice by the hydrodynamics-based procedure. RESULTS: PI was efficiently taken up by hepatocytes which appeared to be viable following the hydrodynamics-based procedure. Pre-expressed GFP in the cytosol was rapidly eliminated from the hepatocytes by a large-volume injection of saline. The profiles of plasma ALT and AST showed a steady decline with the highest values observed immediately after the hydrodynamics-based procedure. These results suggest that the hydrodynamics-based procedure produces a transient increase in the permeability of the cell membrane. The cellular uptake process appeared nonspecific, since simultaneous injection of an excess of empty vector did not affect the transgene expression. Sequential injections of a large volume of pDNA-free saline followed by naked pDNA in a normal volume revealed that the increase in membrane permeability was transient, with a return to normal conditions within 30 min. Transgene expression was observed in hepatocyte cultures isolated 10 min after pDNA delivery and in the liver as early as 10 min after luciferase-expressing RNA delivery, indicating that pDNA delivered immediately by the hydrodynamics-based procedure has the potential to produce successful transgene expression. CONCLUSIONS: These findings suggest that the mechanism for the hydrodynamics-based gene transfer would involve in part the direct cytosolic delivery of pDNA through the cell membrane due to transiently increased permeability.     

Hypothesis: naked plasmid DNA is taken up by cells in vivo by a receptor-mediated process

Following the initial demonstration that intramuscularly-injected naked plasmid DNA (pDNA) is expressed in myofibers, it was shown that pDNA can be used for vaccination purposes. More recent studies have indicated that naked pDNA can also achieve high levels of transgene expression in vivo. This efficiency of naked pDNA expression, especially via intravascular route, is truly astounding. In this prospective review, we examine the possible mechanisms of naked pDNA uptake. The possible mechanisms; (a) large membrane disruption, (b) small membrane pores, and (c) receptor-mediated endocytosis, are considered in turn. Some recent original laboratory data relevant to these hypotheses are also presented.     

ANTI-TUMOR EFFECT OF INTRAVENOUS TNFα GENE DELIVERY NAKED PLASMID DNA USING A HYDRODYNAMICS-BASED PROCEDURE

High levels of foreign gene expression in mouse hepatocytes can be achieved by the rapid injection of a large volume of naked plasmid (pDNA) into animals via the tail vein, the so-called hydrodynamics-based procedure. In this study, we evaluated the efficacy of hydrodynamics-based tumor necrosis factor alpha (TNFα) transfer for tumor treatment, in which the naked pDNA encoding TNFα was administered into the tail vein following an intravenous injection of B16 melanoma cells. The mice treated with TNFα-expressing pDNA displayed a profound reduction in lung metastasis. These results suggest that the hydrodynamics-based transfer of naked pDNA is a convenient and efficient method of TNFα gene therapy against metastatic tumors.     

Anti-tumor effect of intravenous TNFalpha gene delivery naked plasmid DNA using a hydrodynamics-based procedure

High levels of foreign gene expression in mouse hepatocytes can be achieved by the rapid injection of a large volume of naked plasmid (pDNA) into animals via the tail vein, the so-called hydrodynamics-based procedure. In this study, we evaluated the efficacy of hydrodynamics-based tumor necrosis factor alpha (TNFalpha) transfer for tumor treatment, in which the naked pDNA encoding TNFalpha was administered into the tail vein following an intravenous injection of B16 melanoma cells. The mice treated with TNFalpha-expressing pDNA displayed a profound reduction in lung metastasis. These results suggest that the hydrodynamics-based transfer of naked pDNA is a convenient and efficient method of TNFalpha gene therapy against metastatic tumors.     

High volume hydrodynamic injection of plasmid DNA via the hepatic artery results in a high level of gene expression in rat hepatocellular carcinoma induced by diethylnitrosamine

BACKGROUND: Hydrodynamic injection of naked plasmid DNA (pDNA) via the tail vein is a safe and effective method of gene transfer to the liver. However, successful gene transfer has yet to be shown for hepatocellular carcinoma (HCC); therefore, we investigated the feasibility and efficacy of hydrodynamic injection via the tail vein and hepatic artery in a diethylnitrosamine (DEN)-induced HCC model in rats. METHODS: HCC was induced in Sprague-Dawley rats by 100 ppm DEN in drinking water. pCMV-SPORT-beta-galactosidase (beta-gal, 400 microg) was injected (i) via the tail vein in a volume of 0.1 ml/g in 30 s or (ii) via the hepatic artery in a volume of 5 or 10 ml at 1 ml/s, either with or without temporary occlusion of the inferior vena cava (IVC) and portal vein (PV). The liver was harvested 24 h after administration, and beta-gal expression was evaluated with X-gal staining and measurement of enzymatic activity in tissue homogenates. RESULTS: Hydrodynamic injection via the tail vein achieved transgene expression only in non-cancerous tissue (tumor: 0.16 +/- 0.04%, non-tumor: 5.07 +/- 1.66%). Hydrodynamic injection via the hepatic artery was tolerated, but failed to produce efficient transgene expression in tumor and non-tumor cells. On the other hand, concomitant use of temporary IVC/PV occlusion with hydrodynamic injection via the hepatic artery dramatically increased transgene expression in cancer cells, but tumor-selective gene transfer was not achieved with this procedure (tumor: 7.38 +/- 3.66%, non-tumor: 7.77 +/- 1.06%). CONCLUSIONS: High-volume hydrodynamic injection of a pDNA solution via the hepatic artery with IVC/PV occlusion achieved a high level of gene expression in a HCC rat model. This gene transfer technique may have potential in clinical gene therapy for HCC.     

Stimulatory effect of polyethylene glycol (PEG) on gene expression in mouse liver following hydrodynamics-based transfection

BACKGROUND: Rapid intravenous injection of a large volume of plasmid DNA (pDNA), i.e. a transfection procedure based on hydrodynamics, is known to be an efficient and liver-specific method of in vivo gene delivery. However, the gene expression is transient. METHODS: We investigated the effect of addition of polyethylene glycol (PEG) to a solution of naked pDNA (luciferase) on the expression of the gene in mouse liver following transfection by the hydrodynamics-based technique. In addition, the mechanism leading to the enhancement of the gene expression was studied. RESULTS: The addition of 1% (w/v) PEG2000 to the pDNA solution enhanced the resulting gene expression in the liver. Increasing the PEG2000 concentration to more than 1 and up to 10% (w/v) rather diminished the gene expression level. By contrast, increasing the molecular weight of PEG to over 2000 up to 10 000 did not affect the level of gene expression. Histopathological and serum-chemistry examinations indicated that hydrostatic or osmotic pressure increased tissue and hepatocellular damage in a PEG-concentration-dependent manner, and resulted in a decrease in gene expression. Quantitative evaluation showed that the enhanced gene expression resulted from stabilization of the pDNA introduced into the hepatocytes and an enhancement of the transport of intact pDNA to the nucleus. CONCLUSIONS: For most gene therapy applications and gene function studies, sustained expression of the introduced gene(s) is necessary. This simple method to achieve enhanced gene expression in liver may have a great potential for a wide variety of laboratory studies in molecular and cellular biology as well as possibly for future clinical applications in humans.     

Hydrodynamics-based transfection of the liver: entrance into hepatocytes of DNA that causes expression takes place very early after injection

BACKGROUND: The mechanism of gene transfer into hepatocytes by the hydrodynamics-based transfection procedure is not clearly understood. It has been shown that, after a hydrodynamic injection, a large proportion of plasmid DNA remains intact in the liver where it is bound to plasma membrane and suggested that this DNA could be responsible for the efficiency of the transfection. METHODS: We have investigated the problem by giving mice a hydrodynamic injection of isotonic NaCl, followed at different time intervals by a conventional injection of DNA, cold or labelled with (35)S, with cDNA of luciferase as a reporter gene. Then, we determined the consequences of that dual injection on luciferase expression and on DNA uptake by the liver and its intracellular fate. By such experiments, it is possible to establish the time dependency of the induction of liver changes caused by a hydrodynamic injection on the one hand and the expression and DNA uptake and fate on the other. Moreover, some experiments have been performed on primary cultures of hepatocytes isolated after a hydrodynamic injection of DNA. RESULTS: When DNA is given to mice by a conventional injection a few seconds after an hydrodynamic injection of isotonic NaCl, luciferase expression in the liver is considerably lower than that observed after a single hydrodynamic injection of the plasmid. On the other hand, as assessed by the rate of DNA degradation and by centrifugation results obtained after injection of (35)S-DNA, the uptake and the intracellular fate of the bulk of DNA are similar whether DNA is administered by a single hydrodynamic injection or by a conventional injection given up to at least 2 h after a hydrodynamic injection of isotonic NaCl. Hepatocytes isolated a few minutes after a hydrodynamic injection exhibit a maximal expression that does not depend on the large amount of DNA that remains bound to the plasma membrane for a relatively long time. CONCLUSIONS: Our results show that the efficiency of hydrodynamics-based transfection depends on a process that takes place very quickly after injection and is not linked to a delay of DNA degradation and the persistence of a large proportion of DNA bound to hepatocytes of the plasma membrane, strongly suggesting that expression after a hydrodynamic injection is caused by a small proportion of DNA molecules that rapidly enter the cytosol probably by plasma membrane pores generated by the hydrodynamic pressure.     

Successful genetic transduction in vivo into synovium by means of electroporation

This present study aims at establishing a novel in vivo gene delivery system for intra-articular tissues. Plasmid DNA (pDNA) carrying the firefly luciferase or enhanced green fluorescent protein (EGFP) genes as markers was injected into a joint space and electric stimuli were given percutaneously with a pair of electrodes. Injection with naked pDNA alone did not induce any detectable level of luciferase activity, whereas electroporation at 25-500 V/0.7 cm resulted in a significant expression of the marker gene in the synovium. The expression level depended on the voltage, the optimum transfection being achieved at 150 V/0.7 cm. When the Epstein-Barr virus (EBV)-based plasmid vectors harboring the EBV nuclear antigen 1 (EBNA1) gene and oriP sequence were substituted for conventional pDNA, the transfection efficiency was increased approximately 5-10 times. Histological examination of the EGFP gene-transfected joints revealed that the marker gene was expressed in the synovial membrane while other intra-articular tissues such as articular cartilage were negative for the transgene product. Transgene-specific mRNA was demonstrated in synovium but not in other organs as estimated by RT-PCR analysis. The present results strongly suggest that in vivo electroporation is a quite simple, safe, and effective gene delivery method that could be applicable to gene therapy against articular diseases.     

Efficient lipid-mediated transfection of DNA into primary rat hepatocytes

Cationic lipids are an effective means for transfecting nucleic acids into a variety of cell types. Very few of these lipids, however, have been reported to be effective with primary cells. We report on the efficacy of several commercially available cationic lipid reagents to transfect plasmid DNA into primary rat hepatocytes in culture. The reagents tested in this study include TransfectAce, LipofectAmine, Lipofectin, N-[1-(2,3-dioleyloxy)propyl]-n,n,n-trimethylammoniumchloride (DOTMA), (N-[1-(2,3-dioleoyloxy)propyl]-N,N,N-trimethyl-ammonium methylsulfate (DOTAP), and cetyltrimethyl-ammonium bromide/dioleoylphosphatidylethanol-amine (CTAB/DOPE). Electron micrographic (EM) studies indicate that similar size Lipofectin and DOTAP vesicles contain DNA-like material internally and that these vesicles attach to the cell membrane. DOTAP vesicles are multilamellar, appear as clusters, and have a high DNA-to-lipid ratio. Lipofectin vesicles appear to attach to the cell surface as individual vesicles. The EM observations are consistent with current theories on the mechanism of transfection by cationic lipids. While Lipofectin has proven to be effective in transfection studies of primary cells in culture, we have found DOTAP to be a viable alternative. DOTAP yields transfection rates in hepatocytes comparable to DOTMA and Lipofectin, however, at lower concentrations of reagent and at considerably less cost. Optimal conditions for transfecting 5 μg of plasmid DNA with DOTAP were achieved by utilizing multilamellar (vortexed) vesicles at a concentration of 15 μg DOTAP per 2 ml media in 60-mm plates for 2 h transfection time. In this study, DOTAP has proven to be economical, easy to prepare, and very effective in transfecting DNA into primary rat hepatocytes.     

Electroporation enhances reporter gene expression following delivery of naked plasmid DNA to the lung

BACKGROUND: Existing methods of non-viral airway gene transfer suffer from low levels of efficiency. Electroporation has been used to enhance gene transfer in a range of tissues. Here we assess the usefulness of electroporation for enhancing gene transfer in the lungs of mice and sheep. METHODS: Naked plasmid DNA (pDNA) expressing either luciferase or green fluorescent protein (GFP) was delivered to mouse lungs by instillation. Following surgical visualisation, the lungs were directly electroporated and the level and duration of luciferase activity was assessed and cell types that were positive for GFP were identified in lung cryosections. Naked pDNA was nebulised to the sheep lung and electrodes attached to the tip of a bronchoscope were used to electroporate airway segment bifurcations, Luciferase activity was assessed in electroporated and control non-electroporated regions, after 24 h. RESULTS: Following delivery of naked pDNA to the mouse lung, electroporation resulted in up to 400-fold higher luciferase activity than naked pDNA alone when luciferase was under the control of a cytomegalovirus (CMV) promoter. Following delivery of a plasmid containing the human polyubiquitin C (UbC) promoter, electroporation resulted in elevated luciferase activity for at least 28 days. Visualisation of GFP indicated that electroporation resulted in increased GFP detection compared with non-electroporated controls. In the sheep lung electroporation of defined sites in the airways resulted in luciferase activity 100-fold greater than naked pDNA alone. CONCLUSIONS: These results indicate that electroporation can be used to enhance gene transfer in the lungs of mice and sheep without compromising the duration of expression.     

The administration of naked plasmid DNA into the liver induces antitumor innate immunity in a murine liver metastasis model

BACKGROUND: Gene therapy is a promising strategy against advanced cancer; however, the safety of viral vectors and the effectiveness of non-viral vectors have not yet been established. Recently, a hydrodynamics-based procedure was reported to be an effective and safe method to deliver and transduce DNA into the liver. Herein, we propose a strategy for liver metastasis by a hydrodynamics-based procedure to deliver naked non-coding plasmid DNA (pDNA) into the liver as an immunocompetent organ. METHODS AND RESULTS: Mice received a rapid intravenous (i.v.) injection of naked pDNA in a large volume of saline (0.1 ml/g body weight). The single administration of a naked non-coding pDNA by the hydrodynamics-based procedure before tumor cell inoculation strongly suppressed liver metastasis formation. However, the usual i.v. injection (200 microl/body) of the same dose of naked pDNA could not suppress liver metastasis formation. Following the methylation of CpG sequences within the pDNA using CpG methylase, injection of the methylated pDNA by the hydrodynamics-based procedure could not suppress liver metastasis formation. Gadolinium chloride pretreatment did not interfere with this antitumor effect, but anti-asialo GM1 antiserum treatment did. These findings indicated that natural killer (NK) cells, not Kupffer cells, were involved in this antitumor effect. The NK cytotoxic activities of liver mononuclear cells were strongly enhanced after receiving a naked pDNA by the hydrodynamics-based procedure. CONCLUSIONS: These observations suggest that unmethylated CpG motifs in pDNA stimulated immune cells, resulting in the activation of NK cells in the liver to suppress liver metastases in a murine model.     

Rat liver-targeted naked plasmid DNA transfer by tail vein injection

High levels of foreign gene expression in mouse hepatocytes can be achieved by "hydrodynamics-based transfection," the rapid injection of a large volume of a naked deoxyribonucleic acid (DNA) solution into the tail vein. Rats are more tolerant of the frequent phlebotomies required for monitoring blood parameters than mice and, thus, are more suitable for some biomedical research. Recently, we demonstrated that hydrodynamics-based transfection can also be used to deliver naked plasmid DNA into the normal rat, which is more than 10 times larger than the mouse. We performed the tail vein injection using a syringe with a winged needle equipped with an external tube. Injection of a lac Z expression plasmid, pCAGGS-lac Z by this technique resulted in the exclusive detection of beta-galactosidase in the liver. We also injected a rat erythropoietin (Epo) expression plasmid, pCAGGS-Epo (800 microg). Maximal Epo gene expression was achieved when a 25-mL injection volume (approx 100 mL/kg body wt) was transferred within 15 s.     

Novel methods to induce exogenous gene expression in SCNT, parthenogenic and IVF preimplantation bovine embryos

The import of exogenous DNA (eDNA) from the cytoplasm to the nucleus represents a key intracellular obstacle for efficient gene delivery in mammalian cells. In this study, cumulus cells or oolemma vesicles previously incubated with eDNA, and naked eDNA were injected into the cytoplasm of MII oocytes to evaluate their efficiency for eDNA expressing bovine embryo production. Our study evaluated the potential of short time co-incubation (5 min) of eDNA with; (1) cumulus cells, to be used as donor cells for SCNT and (2) oolemma vesicles (vesicles) to produce parthenogenic transgene expressing embryos. In addition, we included a group consisting of the injection of eDNA alone (plasmid) followed by parthenogenic activation. Two different pCX-EGFP plasmid concentrations (50 and 500 ng/μl) were employed. The results showed that embryos produced by SCNT and by vesicle injection assisted by chemical activation were able to express the eDNA in higher rates than embryos injected with plasmid alone. The lower plasmid concentration allowed the highest development rates in all groups. Using confocal microscopy, we analyzed the interaction of FITC- labeled eDNA with cumulus cells and vesicles as well as oocytes injected with labeled plasmid alone. Our images demonstrated that eDNA interacted with cumulus cells and vesicles, resulting an increase in its expression efficiency. In contrast, oocytes injected with DNA alone did not show signs of transgene accumulation, and their eDNA expression rates were lower. In a further experiment, we evaluated if transgene-expressing embryos could be produced by means of vesicle injection followed by IVF. The lower plasmid concentration (50 ng/μl) injected after IVF, produced the best results. Preliminary FISH analysis indicated detectable integration events in 1/5 of SCNT blastocysts treated. Our studies demonstrate for the first time that short term transgene co-incubation with somatic cells can produce transgene-expressing mammalian SCNT embryos and also that parthenogenic, eDNA- expressing embryos can be obtained by injection of vesicles or eDNA alone. Moreover, eDNA-expressing embryos can be also obtained by cytoplasmic injection vesicles in IVF zygotes, simplifying the traditional IVF pronuclear injection technique.     

Transient non-viral cutaneous gene delivery in burn wounds

BACKGROUND: Gene transfer to burn wounds could present an alternative to conventional and often insufficient topical and systemic application of therapeutic agents to aid in wound healing. The goals of this study were to assess and optimize the potential of transient non-viral gene delivery to burn wounds. METHODS: HaCaT cells were transfected with luciferase or beta-galactosidase transgene using either pure plasmid DNA (pDNA) or complexed with Lipofectamine 2000, FuGENE6, or DOTAP-Chol. Expression was determined by bioluminescence and fluorescence. Forty male Sprague-Dawley rats received naked pDNA, lipoplexes, or carrier control intradermally into either unburned skin, superficial, partial, or full-thickness scald burn. Animals were sacrificed after 24 h, 48 h, or 7 days, and transgene expression was assessed. RESULTS: Gene transfer to HaCaT cells showed the overall highest expression for DOTAP/Chol (77.85 ng luciferase/mg protein), followed by Lipofectamine 2000 (33.14 ng luciferase/mg protein). pDNA-derived gene transfer to superficial burn wounds showed the highest expression among burn groups (0.77 ng luciferase/mg protein). However, lipoplex-derived gene transfer to superficial burns and unburned skin failed to show higher expression. CONCLUSIONS: Lipofectamine 2000 and DOTAP/Chol lipoplex showed significantly enhanced gene transfer, whereas no transfection was detectable for naked DNA in vitro. In contrast to the in vitro study, naked DNA was the only agent with which gene delivery was successful in experimental burn wounds. These findings highlight the limited predictability of in vitro analysis for gene delivery as a therapeutic approach.     

Studies on aerosol delivery of plasmid DNA using a mesh nebulizer

Aerosol delivery of plasmid DNA therapeutic solutions is promising for the treatment of respiratory diseases. However, it poses challenges, most significantly the need to protect the delicate supercoiled (sc) structure of plasmid during aerosolization. Nebulizers for liquid aerosolization using meshes appear a better method for delivery than conventional jet and ultrasonic nebulizers. This paper explores their application to the delivery of plasmid DNA. A computational fluid dynamics model of the dynamics of fluid flow through the nozzle of the MicroAIR mesh nebulizer indicated high strain rates (>10(5) s(-1)) near the nozzle exit capable of causing damage to the shear-sensitive plasmid DNA. Knowledge of the strain rates predicted using CFD and molecule size determined using atomic force microscopy (AFM) enabled estimation of the hydrodynamic force and whether damage of shear-sensitive therapeutics was likely. Plasmids of size 5.7 and 20 kb were aerosolized in the mesh nebulizer. The sc structure of the 5.7-kb plasmid was successfully delivered without damage, while aerosolization of the 20-kb plasmid led to disintegration of the pDNA sc structure as observed in AFM. Subsequent formulation of the sc 20-kb plasmid with PEI resulted in successful aerosol delivery. The maximum hydrodynamic forces computed for the aerosolization of structures of the size of 5.7-kb and PEI formulated 20-kb plasmids were less than the forces reported to damage the structure of double-stranded DNA. A combination of CFD analysis and structure analysis may be used to predict successful aerosol delivery in such a mesh nebulizer.     

In vivo Site-Specific Transfection of Naked Plasmid DNA and siRNAs in Mice by Using a Tissue Suction Device

We have developed an in vivo transfection method for naked plasmid DNA (pDNA) and siRNA in mice by using a tissue suction device. The target tissue was suctioned by a device made of polydimethylsiloxane (PDMS) following the intravenous injection of naked pDNA or siRNA. Transfection of pDNA encoding luciferase was achieved by the suction of the kidney, liver, spleen, and heart, but not the duodenum, skeletal muscle, or stomach. Luciferase expression was specifically observed at the suctioned region of the tissue, and the highest luciferase expression was detected at the surface of the tissue (0.12±0.03 ng/mg protein in mice liver). Luciferase expression levels in the whole liver increased linearly with an increase in the number of times the liver was suctioned. Transfection of siRNA targeting glyceraldehyde 3-phosphate dehydrogenase (GAPDH) gene significantly suppressed the expression of GAPDH mRNA in the liver. Histological analysis shows that severe damage was not observed in the suctioned livers. Since the suction device can be mounted onto the head of the endoscope, this method is a minimally invasive. These results indicate that the in vivo transfection method developed in this study will be a viable approach for biological research and therapies using nucleic acids.     

Mechanism of naked DNA clearance after intravenous injection

BACKGROUND: Injection of naked DNA has been viewed as a safer alternative to current gene delivery systems; however, the rate of clearance from the circulation has been a constant barrier in developing these methods. Naked DNA after intravenous (i.v.) injection will be taken up by the liver and depredated by serum nucleases. MATERIALS AND METHODS: Our study examines the mechanisms involved in clearance of naked DNA by each compartment, the blood and the liver, in an in vivo mouse model. Confocal microscopy and transmission electron microscopy were employed to identify the type of cells taking up DNA and the barrier to DNA access to hepatocytes, respectively. RESULTS: Our data showed the liver could take up over 50% of 5 microg perfused pDNA, with a maximum 25 microg of pDNA during a single pass, and a slower clearance rate compared to that of liver uptake. It was demonstrated that naked DNA is primarily taken up by the liver endothelial cells and this endothelial barrier to transfection could be overcome by manually massaging the liver, which enlarges the fenestrae. CONCLUSIONS: This study clarifies the mechanism by which naked DNA is eliminated from the circulation after i.v. injection, focusing on the role of both the liver and blood compartments in vivo (i.e. mouse). With this knowledge, we can more clearly understand the mechanism of naked DNA clearance and develop more efficient strategies for DNA transfer in vivo.     

Microencapsulation of nanoparticulate complexes of DNA with cationic lipids and polymers in pectin beads for targeted gene delivery

Nanoparticulate complexes of plasmid DNA (pDNA) with cationic liposomes/polymer, of approx 200 nm diameter, were encapsulated with a high degree of efficiency within calcium pectinate gel beads. Electron microscopy showed the DNA nanocomplexes to be evenly distributed throughout the gel matrix. Controlled release of pDNA-lipid nanocomplexes was achieved by the action of pectinase enzymes, whereas release of naked and polymer-complexed DNA was found to be more greatly influenced by the swelling behavior of the polysaccharide matrices in buffer alone. Physical degradation of pDNA within pectin beads was found to be accelerated during bead drying, most probably as a result of shear forces generated within the gel matrices by the evaporation of water. Plasmid complexation with cationic liposomes provided a greater degree of protection for the DNA during bead drying than complexation with cationic polymer, and was shown to successfully transfect cultured cells after release from the beads, via the action of pectinase. Observations concerning the physical stability of nanocomplexed pDNA, and its encapsulation within and release from pectin gel beads, are discussed with reference to the electrostatic interactions existing between the various components.