Single intravenous injection of naked plasmid DNA encoding erythropoietin provides neuroprotection in hypoxia-ischemia rats

Hypoxic-ischemic (H-I) encephalopathy is a major contributor to morbidity and mortality in infants and children. To delineate the nature and mechanism(s) of neuroprotection via erythropoietin (EPO) gene therapy, we evaluated the effects of single intravenous injection of naked plasmid DNA encoding EPO in H-I infant rats. Single administration of naked plasmid containing EPO cDNA driven under cytomegalovirus promoter (pCMV-EPO) by rapid injection via the tail vein produced a remarkable level of human EPO protein in the circulation, peaking at one day and lasting for 14 days after injection. There were significant improvements of water maze task in H-I rats after EPO gene therapy. Our data showed that the mechanisms of EPO gene therapy were rescue of CA1 neurons from lethal H-I injury, prevention of neuronal apoptosis in CA1 region, and decrease of glial activation in corpus callosum. This could be the first report of successful treatment of H-I injury by a single intravenous infusion of EPO gene.     

Conditional gene modification in mouse liver using hydrodynamic delivery of plasmid DNA encoding Cre recombinase

The success of Cre-mediated conditional gene targeting in liver of mice has until now depended on the generation of Cre recombinase transgenic mice or on viral-mediated transduction. Here, we sought to establish the feasibility of using hydrodynamic gene delivery of Cre recombinase into liver, using a ROSA26 EGFP mouse. The expression of EGFP and beta-galactosidase was exclusively detected in the liver of mice treated with hydrodynamic gene delivery of Cre recombinase, as assessed with fluorescence microscopy and X-Gal staining, respectively; Southern blotting also showed that Cre mediated recombination occurred specifically in the liver and not in other organs. The Cre mediated recombination reached about 61% of hepatocytes of mouse after repeated injection, as analyzed by flow cytometry. These results demonstrate that Cre recombinase can be transferred to the liver of mice through a simple hydrodynamic gene-delivery approach and can mediate efficient recombination in hepatocytes. Thus, hydrodynamic gene delivery of the Cre recombinase provides a valuable approach for Cre-loxP-mediated conditional gene modification in the liver of mice.     

Transplacental genetic immunization after intravenous delivery of plasmid DNA to pregnant mice.

A number of factors influence the development of tolerance, including the nature, concentration, and mode of Ag presentation to the immune system, as well as the age of the host. The studies were conducted to determine whether immunizing pregnant mice with liposome-encapsulated DNA vaccines had an effect on the immune status of their offspring. Two different plasmids (encoding Ags from HIV-1 and influenza virus) were administered i.v. to pregnant mice. We examined the uptake of plasmid DNA by the fetuses until the 21st postcoital day, but little such transfer occurred in early pregnancy. At 9.5 days postconception with cationic liposomes, injected plasmid was present in the tissues of the fetus, consistent with transplacental transfer. When the offspring of vaccinated dams were immunized with DNA vaccine, they mounted stronger Ag-specific immune responses than controls, and were protected against challenge by homologous influenza virus after vaccination. Moreover, such immune responses were strong in the offspring of mothers injected with DNA plasmid 9.5 days after coitus. These results suggest that DNA-vaccinated mothers confer the Ag-specific immunity to their progeny.     

Effects of leptin gene expression in mice in vivo by electroporation and hydrodynamics-based gene delivery

In vivo electroporation and hydrodynamics-based gene delivery were utilized to test the effect of leptin gene transfer on food intake, and body and fat weights of mice. Gene transfer of pVRmob by electroporation caused a significant reduction in body weight compared with the control counterpart (p<0.05), although a lesser effect was found in food intake, and the weights of interscapular brown and epididymal fat by electroporation. As might be expected, the hydrodynamics-based transfection method significantly reduced body weight over 1 week post-transfection (p<0.05). Furthermore, epididymal fat was decreased by 50% at 1 week after gene transfer (p<0.001). These results suggest that both electroporation and hydrodynamics-based gene delivery may be effective approaches for systemic delivery of recombinant leptin to the central nervous system, and that the efficiency of gene transfer in hydrodynamics-based gene delivery was markedly higher than that in electroporation at least within the first week after transfection.     

Dendritic cell delivery of plasmid DNA

Positive human clinical data using biolistic-mediated gene transfer (i.e., gene gun) to administer a nucleic acid-based Hepatitis B vaccine has validated genetic immunization as an effective clinical vaccine modality. Although the precise mechanism of action has yet to be determined, preclinical studies using jet injection have indicated that direct targeting of resident antigen presenting cells (Langerhan's cells) in the skin as the primary immunological driving force for the potent and long-lived immune response. Moreover, positive results with topical delivery of genetic vaccines and ex vivo loading of dendritic cells with antigen has strengthened the movement toward directly targeting antigen presenting cells as a means to amplify, control, and mediate the immunological consequences of prophylactic and/or therapeutic genetic vaccines. Despite these encouraging results with the gene gun, it is unclear whether this technology will translate into commercially available vaccines due to potential product development barriers such as cost and convenience. It is clear that safety concerns in using genetic approaches to treat and prevent disease have highlighted the need for strict product requirements for genetic vaccines. A plausible strategy to meet these requirements is to combine controlled plasmid delivery systems with tissue-specific gene expression systems.     

Controlled delivery of plasmid DNA and siRNA to intracellular targets using ketalized polyethylenimine

A new polyethylenimine (PEI)-derived biodegradable polymer was synthesized as a nonviral gene carrier. Branches of PEI were ketalized, and capabilities of nucleic acid condensation and delivery efficiency of the modified polymers were compared with ones of unketalized PEI. Ketalized PEI was able to efficiently compact both plasmid DNA and siRNA into nucleic acids/ketalized PEI polyplexes with a range of 80-200 nm in diameter. Nucleic acids were efficiently dissociated from the polyplexes made of ketalized PEI upon hydrolysis. In vitro study also demonstrated that ketalization enhanced transfection efficiency of the polyplexes while reducing cytotoxicity, even at high N/ P ratios. Interestingly, transfection efficiency was found to be inversely proportional to molecular weights of ketalized PEI, while RNA interference was observed in the opposite way. This study implies that selective delivery of plasmid DNA and siRNA to the nucleus and the cytoplasm can be achieved by tailoring the structures of polymeric gene carriers.     

Optimization of naked DNA delivery for interferon subtype immunotherapy in cytomegalovirus infection

Type I interferon (IFN) gene therapy modulates the immune response leading to inflammatory heart disease following cytomegalovirus (CMV) infection in a murine model of post-viral myocarditis. Efficacy of different immunisation protocols for the IFN constructs was influenced by the dose of DNA, subtype choice, combination use, pre-medication, and timing of DNA administration. Optimal efficacy was found with bupivacaine treatment prior to DNA inoculation of 200μgIFN DNA 14 days prior to virus challenge. Maximal antiviral and antimyocarditic effects were achieved with this vaccination schedule. Furthermore, inoculation of synergistic IFN subtypes demonstrated enhanced efficacy when delivered either alone or with CMVgB DNA vaccination in the CMV model. Thus naked DNA delivery of IFN provides an avenue of immunotherapy for regulating herpesvirus-induced diseases. Published: February 17, 2003     

A simple procedure for large-scale purification of plasmid DNA

We report a simple, rapid and reliable procedure for large-scale purification of plasmid DNA from non-amplified bacterial cultures. It is a modification of the boiling method of Holmes and Quigley [Anal. Biochem. 114 (1981) 193-197] and involves gel-filtration chromatography using Sephacryl S-1000 for final purification of plasmid DNA. This method does not require CsCl gradients and the recovered plasmids are free of RNA and chromosomal DNA, are supercoiled, retain their biological activity, and are suitable for restriction analysis.     

Polyplexes assembled with internally quaternized PAMAM-OH dendrimer and plasmid DNA have a neutral surface and gene delivery potency

Interior tertiary amine groups of PAMAM-OH dendrimers (hydroxyl-terminated polyamidoamine, PAMAM) were modified by methylation to make these polymers have a more cationic character, which enabled electrostatic interaction between PAMAM-OH and plasmid DNA. A methylation reaction was dose-dependent, producing internally quaternized PAMAM-OH (QPAMAM-OH), thereby making tertiary amine/quaternary amine ratio adjustment possible. More highly condensed particles of plasmid DNA were formed as the degree of quaternization increased, whereas unmodified polymer (PAMAM-OH) could not. The location of positive charges in the internal position of QPAMAM-OH resulted in the formation of neutral polyplexes in which zeta potential leveled off near the zero value even at high charge ratios (+/-) of 10. A light scattering experiment showed that the polyplex formed by QPAMAM-OH was very small with the size of 53.3 nm at the optimum condition. QPAMAM-OH/DNA polyplexes were round-shaped with the more compact and small particles formed as the charge ratio increased. QPAMAM-OH showed much reduced cytotoxicity compared with starburst PAMAM and branched polyethyleneimine (PEI) in which shielding of interior positive charges by surface hydroxyls might be the reason for this favorable result. These results suggest that QPAMAM-OH could be a promising tool as a nonviral vector both by itself and in conjugated form with targeting ligands.     

Dual bio-responsive gene delivery via reducible poly(amido amine) and survivin-inducible plasmid DNA

A bioreducible poly(amido amine) (SS-PAA) gene carrier, known as poly (amido-butanol) (pABOL), was used to transfect a variety of cancer and non-cancer cell lines. To obtain cancer-specific transgene expression for therapeutic efficiency in cancer treatment, we constructed survivin-inducible plasmid DNA expressing the soluble VEGF receptor, sFlt-1, downstream of the survivin promoter (pSUR-sFlt-1). Cancer-specific expression of sFlt-1 was observed in the mouse renal carcinoma (RENCA) cell line. pABOL enhanced the efficiency of gene delivery compared to traditional carriers used in the past. Thus, a dual bio-responsive gene delivery system was developed by using bioreducible p(ABOL) for enhanced intracellular gene delivery and survivin-inducible gene expression system (pSUR-sFlt-1 or pSUR-Luc reporter gene) that demonstrates increased gene expression in cancer that has advantages over current gene delivery systems.     

Tissue distribution of a plasmid DNA encoding Hsp65 gene is dependent on the dose administered through intramuscular delivery

In order to assess a new strategy of DNA vaccine for a more complete understanding of its action in immune response, it is important to determine the in vivo biodistribution fate and antigen expression. In previous studies, our group focused on the prophylactic and therapeutic use of a plasmid DNA encoding the Mycobacterium leprae 65-kDa heat shock protein (Hsp65) and achieved an efficient immune response induction as well as protection against virulent M. tuberculosis challenge. In the present study, we examined in vivo tissue distribution of naked DNA-Hsp65 vaccine, the Hsp65 message, genome integration and methylation status of plasmid DNA. The DNA-Hsp65 was detectable in several tissue types, indicating that DNA-Hsp65 disseminates widely throughout the body. The biodistribution was dose-dependent. In contrast, RT-PCR detected the Hsp65 message for at least 15 days in muscle or liver tissue from immunized mice. We also analyzed the methylation status and integration of the injected plasmid DNA into the host cellular genome. The bacterial methylation pattern persisted for at least 6 months, indicating that the plasmid DNA-Hsp65 does not replicate in mammalian tissue, and Southern blot analysis showed that plasmid DNA was not integrated. These results have important implications for the use of DNA-Hsp65 vaccine in a clinical setting and open new perspectives for DNA vaccines and new considerations about the inoculation site and delivery system.     

Efficient in vivo gene delivery by the negatively charged complexes of cationic liposomes and plasmid DNA

We examined changes in zeta potential (the surface charge density, zeta) of the complexes of liposome (nmol)/DNA (microg) (L/D) formed in water at three different ratios (L/D=1, 10 and 20) by changing the ionic strength or pH to find an optimum formulation for in vivo gene delivery. At high DNA concentrations, zeta of the complexes formed in water at L/D=10 was significantly lowered by adding NaCl (zeta=+8.44+/-3.1 to -27.6+/-3.5 mV) or increasing pH from 5 (zeta=+15.3+/-1.0) to 9 (zeta=-22.5+/-2.5 mV). However, the positively charged complexes formed at L/D=20 (zeta=+6.2+/-3.5 mV) became negative as NaCl was added at alkaline pH as observed in medium (zeta=-19.7+/-9.9 mV). Thus, the complexes formed in water under the optimum condition were stable and largely negatively charged at L/D=1 (zeta=-58.1+/-3.9 mV), unstable and slightly positively charged at L/D=10 (zeta=+8.44+/-3.7 mV), and unstable and largely positively charged at L/D=20 (zeta=+24.3+/-3.6 mV). The negatively charged complexes efficiently delivered DNA into both solid and ascitic tumor cells. However, the positively charged complexes were very poor in delivering DNA into solid tumors, yet were efficient in delivering DNA into ascitic tumors grown in the peritoneum regardless of complex size. This slightly lower gene transfer efficiency of the negatively charged complexes can be as efficient as the positively charged ones when an injection is repeated (at least two injections), which is the most common case for therapy regimes. The results indicate that optimum in vivo lipofection may depend on the site of tumor growth.     

A simple procedure for maximum yield of high-quality plasmid DNA

We have established a simple procedure for the rapid isolation of high-quality plasmid DNA suitable for various molecular techniques and provided a step-by-step protocol. The DNA samples isolated by this procedure have been used successfully for double-stranded DNA sequencing, restriction enzyme mapping, subcloning, in vitro mutagenesis, generation of deletion clones and so on. The procedure is highly reproducible, and superior quality DNA can be obtained without the use of phenol, chloroform or other organic solvents.     

Enhancement of immune responses by DNA vaccination through targeted gene delivery using mannosylated cationic liposome formulations following intravenous administration in mice

The present study investigated the potency of the mannosylated cationic liposomes (Man liposomes) that we have developed in novel DNA vaccine carrier. Ovalbumin (OVA) was selected as a model antigen for vaccination; accordingly, OVA-encoding pDNA (pCMV-OVA) was constructed to evaluate DNA vaccination. The potency of the Man liposome/pCMV-OVA complex was compared with naked pCMV-OVA and that complexed with DC-Chol liposomes. In cultured mouse peritoneal macrophages, MHC class I-restricted antigen presentation of the Man liposome/pCMV-OVA complex was significantly higher than that of naked pCMV-OVA and that complexed with DC-Chol liposomes. After intravenous administration, OVA mRNA expression and MHC class I-restricted antigen presentation on CD11c+ cells and inflammatory cytokines, such as TNF-alpha, IL-12, and IFN-gamma, that can enhance the Th1 response of the Man liposome/pCMV-OVA complex were higher than that of naked pCMV-OVA and that complexed with DC-Chol liposomes. Also, the spleen cells from mice immunized by intravenous administration of the Man liposome/pCMV-OVA complex showed the highest proliferation response and IFN-gamma secretion. These findings suggest that the targeted delivery of DNA vaccine by Man liposomes is a potent vaccination method for DNA vaccine therapy.     

Systemic administration of naked DNA: gene transfer to skeletal muscle

Skeletal muscle is a promising target tissue for the gene therapy of both muscle and non-muscle disorders. Gene transfer into muscle tissue can produce a variety of physiologically active proteins and may ultimately be applied to the treatment of many diseases. A variety of methods have been studied to transfer genes into skeletal muscle, including viral and non-viral vectors. In this review, we discuss recent developments in the non-viral delivery of genes to muscles.