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Hamad MIK, Daoud S, Petrova P, Rabaya O, Jbara A, Melliti N, Stichmann S, Reiss G, Herz J, Förster E. Biolistic transfection and expression analysis of acute cortical slices. J Neurosci Methods 2020; 337:108666. [PMID: 32119875 DOI: 10.1016/j.jneumeth.2020.108666] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.2] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/14/2019] [Revised: 02/21/2020] [Accepted: 02/27/2020] [Indexed: 12/28/2022]
Abstract
BACKGROUND Biolistic gene gun transfection has been used to transfect organotypic cultures (OTCs) or dissociated cultures in vitro. Here, we modified this technique to allow successful transfection of acute brain slices, followed by measurement of neuronal activity within a few hours. NEW METHOD We established biolistic transfection of murine acute cortical slices to measure calcium signals. Acute slices are mounted on plasma/thrombin coagulate and transfected with a calcium sensor. Imaging can be performed within 4 h post transfection without affecting cell viability. RESULTS Four hours after GCaMP6s transfection, acute slices display remarkable fluorescent protein expression level allowing to study spontaneous activity and receptor pharmacology. While optimal gas pressure (150 psi) and gold particle size used (1 μm) confirm previously published protocols, the amount of 5 μg DNA was found to be optimal for particle coating. COMPARISON WITH EXISTING METHODS The major advantage of this technique is the rapid disposition of acute slices for calcium imaging. No transgenic GECI expressing animals or OTC for long periods are required. In acute slices, network interaction and connectivity are preserved. The method allows to obtain physiological readouts within 4 h, before functional tissue modifications might come into effect. Limitations of this technique are random transfection, low expression efficiency when using specific promotors, and preclusion or genetic manipulations that require a prolonged time before physiological changes become measurable, such as expression of recombinant proteins that require transport to distant subcellular localizations. CONCLUSION The method is optimal for short-time investigation of calcium signals in acute slices.
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Affiliation(s)
- Mohammad I K Hamad
- Institute for Anatomy and Clinical Morphology, School of Medicine, Faculty of Health, University of Witten/Herdecke, Witten, Germany; Department of Neuroanatomy and Molecular Brain Research, Ruhr University Bochum, Medical Faculty, Bochum, Germany.
| | - Solieman Daoud
- Department of Neuroanatomy and Molecular Brain Research, Ruhr University Bochum, Medical Faculty, Bochum, Germany
| | - Petya Petrova
- Department of Neuroanatomy and Molecular Brain Research, Ruhr University Bochum, Medical Faculty, Bochum, Germany
| | - Obada Rabaya
- Department of Neuroanatomy and Molecular Brain Research, Ruhr University Bochum, Medical Faculty, Bochum, Germany
| | - Abdalrahim Jbara
- Department of Neuroanatomy and Molecular Brain Research, Ruhr University Bochum, Medical Faculty, Bochum, Germany
| | - Nesrine Melliti
- Institute for Anatomy and Clinical Morphology, School of Medicine, Faculty of Health, University of Witten/Herdecke, Witten, Germany
| | - Sarah Stichmann
- Department of Neuroanatomy and Molecular Brain Research, Ruhr University Bochum, Medical Faculty, Bochum, Germany
| | - Gebhard Reiss
- Institute for Anatomy and Clinical Morphology, School of Medicine, Faculty of Health, University of Witten/Herdecke, Witten, Germany
| | - Joachim Herz
- Departments of Molecular Genetics, Neuroscience, Neurology and Neurotherapeutics, Center for Translational Neurodegeneration Research, University of Texas Southwestern Medical Center, Dallas, TX, USA
| | - Eckart Förster
- Department of Neuroanatomy and Molecular Brain Research, Ruhr University Bochum, Medical Faculty, Bochum, Germany
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Abstract
Understanding the rules of synapse dynamics in the context of development, learning, and nervous system disorders is an important part of several fields of neuroscience. Despite significant methodological advances, observations of structural dynamics of synapses still present a significant experimental challenge. In this chapter we describe a set of techniques that allow repetitive observations of synaptic structures in vitro in organotypic cultures of rodent hippocampus. We describe culturing of slices, transfection with reporter-carrying plasmids, repetitive imaging of dendritic spines with confocal laser scanning microscopy and analysis of spine morphology dynamics.
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Zhang D, Rielly CD, Das DB. Microneedle-assisted microparticle delivery by gene guns: experiments and modeling on the effects of particle characteristics. Drug Deliv 2014; 22:335-50. [PMID: 24524342 DOI: 10.3109/10717544.2014.887158] [Citation(s) in RCA: 9] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/16/2022] Open
Abstract
Microneedles (MNs) have been shown to enhance the penetration depths of microparticles delivered by gene gun. This study aims to investigate the penetration of model microparticle materials, namely, tungsten (<1 μm diameter) and stainless steel (18 and 30 μm diameters) into a skin mimicking agarose gel to determine the effects of particle characteristics (mainly particle size). A number of experiments have been processed to analyze the passage percentage and the penetration depth of these microparticles in relation to the operating pressures and MN lengths. A comparison between the stainless steel and tungsten microparticles has been discussed, e.g. passage percentage, penetration depth. The passage percentage of tungsten microparticles is found to be less than the stainless steel. It is worth mentioning that the tungsten microparticles present unfavourable results which show that they cannot penetrate into the skin mimicking agarose gel without the help of MN due to insufficient momentum due to the smaller particle size. This condition does not occur for stainless steel microparticles. In order to further understand the penetration of the microparticles, a mathematical model has been built based on the experimental set up. The penetration depth of the microparticles is analyzed in relation to the size, operating pressure and MN length for conditions that cannot be obtained in the experiments. In addition, the penetration depth difference between stainless steel and tungsten microparticles is studied using the developed model to further understand the effect of an increased particle density and size on the penetration depth.
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Affiliation(s)
- Dongwei Zhang
- Department of Chemical Engineering, Loughborough University , Loughborough , UK
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Zhang D, Das DB, Rielly CD. Microneedle assisted micro-particle delivery from gene guns: experiments using skin-mimicking agarose gel. J Pharm Sci 2014; 103:613-27. [PMID: 24399616 DOI: 10.1002/jps.23835] [Citation(s) in RCA: 52] [Impact Index Per Article: 4.7] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/13/2013] [Revised: 11/11/2013] [Accepted: 12/10/2013] [Indexed: 11/07/2022]
Abstract
A set of laboratory experiments has been carried out to determine if micro-needles (MNs) can enhance penetration depths of high-speed micro-particles delivered by a type of gene gun. The micro-particles were fired into a model target material, agarose gel, which was prepared to mimic the viscoelastic properties of porcine skin. The agarose gel was chosen as a model target as it can be prepared as a homogeneous and transparent medium with controllable and reproducible properties allowing accurate determination of penetration depths. Insertions of various MNs into gels have been analysed to show that the length of the holes increases with an increase in the agarose concentration. The penetration depths of micro-particle were analysed in relation to a number of variables, namely the operating pressure, the particle size, the size of a mesh used for particle separation and the MN dimensions. The results suggest that the penetration depths increase with an increase of the mesh pore size, because of the passage of large agglomerates. As these particles seem to damage the target surface, then smaller mesh sizes are recommended; here, a mesh with a pore size of 178 μm was used for the majority of the experiments. The operating pressure provides a positive effect on the penetration depth, that is it increases as pressure is increased. Further, as expected, an application of MNs maximises the micro-particle penetration depth. The maximum penetration depth is found to increase as the lengths of the MNs increase, for example it is found to be 1272 ± 42, 1009 ± 49 and 656 ± 85 μm at 4.5 bar pressure for spherical micro-particles of 18 ± 7 μm diameter when we used MNs of 1500, 1200 and 750 μm length, respectively.
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Affiliation(s)
- Dongwei Zhang
- Department of Chemical Engineering, Loughborough University, Loughborough, LE113TU, UK
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Zhang D, Das DB, Rielly CD. Potential of microneedle-assisted micro-particle delivery by gene guns: a review. Drug Deliv 2013; 21:571-87. [PMID: 24313864 DOI: 10.3109/10717544.2013.864345] [Citation(s) in RCA: 36] [Impact Index Per Article: 3.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/21/2022] Open
Abstract
CONTEXT Gene guns have been used to deliver deoxyribonucleic acid (DNA) loaded micro-particle and breach the muscle tissue to target cells of interest to achieve gene transfection. OBJECTIVE This article aims to discuss the potential of microneedle (MN) assisted micro-particle delivery from gene guns, with a view to reducing tissue damage. METHODS Using a range of sources, the main gene guns for micro-particle delivery are reviewed along with the primary features of their technology, e.g. their design configurations, the material selection of the micro-particle, the driving gas type and pressure. Depending on the gene gun system, the achieved penetration depths in the skin are discussed as a function of the gas pressure, the type of the gene gun system and particle size, velocity and density. The concept of MN-assisted micro-particles delivery which consists of three stages (namely, acceleration, separation and decoration stage) is discussed. In this method, solid MNs are inserted into the skin to penetrate the epidermis/dermis layer and create holes for particle injection. Several designs of MN array are discussed and the insertion mechanism is explored, as it determines the feasibility of the MN-based system for particle transfer. RESULTS This review suggests that one of the problems of gene guns is that they need high operating pressures, which may result in direct or indirect tissue/cells damage. MNs seem to be a promising method which if combined with the gene guns may reduce the operating pressures for these devices and reduce tissue/cell damages. CONCLUSIONS There is sufficient potential for MN-assisted particle delivery systems.
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Affiliation(s)
- Dongwei Zhang
- Department of Chemical Engineering, Loughborough University , Loughborough, Leicestershire , UK
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Li S, Tang Z, Yu H, Li W, Jiang Y, Wang Y, An W. Administration of naked plasmid encoding hepatic stimulator substance by hydrodynamic tail vein injection protects mice from hepatic failure by suppressing the mitochondrial permeability transition. J Pharmacol Exp Ther 2011; 338:750-7. [PMID: 21613410 DOI: 10.1124/jpet.111.181305] [Citation(s) in RCA: 15] [Impact Index Per Article: 1.1] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/16/2022] Open
Abstract
Acute liver failure is a devastating illness of various causes with considerable mortality. Hepatic stimulator substance (HSS) has been suggested for use as a protective agent against acute hepatic injury induced by chemical poisons because it has a variety of biological activities. However, the mechanism whereby HSS protects against hepatotoxins is poorly understood. In this study, we established a hepatic gene transfer system via hydrodynamic tail vein injection to deliver a naked plasmid containing the human HSS gene (hHSS) and analyzed HSS-mediated protection of the liver during fulminant hepatic failure (FHF) induced by D-galactosamine (D-gal) and lipopolysaccharide (LPS). The results showed that the reporter gene, enhanced green fluorescent protein, was efficiently expressed in the liver of BALB/c mice. Hydrodynamic-based transfection of hHSS yielded a 70% survival rate compared with 36.7% for the control group at 24 h after D-gal/LPS treatment. In addition, hHSS expression preserved liver morphology and function. It is noteworthy that hHSS hydrodynamic-based transfer ameliorated indices of the mitochondrial permeability transition (MPT) resulting from the toxic effects of d-gal/LPS on the liver such as mitochondrial swelling, mitochondrial transmembrane potential disruption, and cytochrome c translocation. Furthermore, mitochondrial morphology and ATP levels were maintained in hHSS-administered mice. HSS-mediated protection was similar to that observed with the MPT inhibitor N-methyl-4-isoleucine-cyclosporin (NIM811), indicating a possible role for HSS in the regulation of MPT. In conclusion, a single dose of hHSS plasmid protected mice from FHF, and this hepatoprotective effect seemed to correlate with the inhibition of MPT.
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Affiliation(s)
- Shenglan Li
- Department of Cell Biology and Municipal Laboratory for Liver Protection and Regulation of Regeneration, Capital Medical University, Beijing, China
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Zeng P, Xu Y, Zeng C, Ren H, Peng M. Chitosan-modified poly(d,l-lactide-co-glycolide) nanospheres for plasmid DNA delivery and HBV gene-silencing. Int J Pharm 2011; 415:259-66. [DOI: 10.1016/j.ijpharm.2011.05.053] [Citation(s) in RCA: 38] [Impact Index Per Article: 2.7] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/05/2011] [Revised: 04/26/2011] [Accepted: 05/20/2011] [Indexed: 12/18/2022]
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Xia J, Martinez A, Daniell H, Ebert SN. Evaluation of biolistic gene transfer methods in vivo using non-invasive bioluminescent imaging techniques. BMC Biotechnol 2011; 11:62. [PMID: 21635760 PMCID: PMC3125329 DOI: 10.1186/1472-6750-11-62] [Citation(s) in RCA: 13] [Impact Index Per Article: 0.9] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/04/2011] [Accepted: 06/02/2011] [Indexed: 01/01/2023] Open
Abstract
Background Gene therapy continues to hold great potential for treating many different types of disease and dysfunction. Safe and efficient techniques for gene transfer and expression in vivo are needed to enable gene therapeutic strategies to be effective in patients. Currently, the most commonly used methods employ replication-defective viral vectors for gene transfer, while physical gene transfer methods such as biolistic-mediated ("gene-gun") delivery to target tissues have not been as extensively explored. In the present study, we evaluated the efficacy of biolistic gene transfer techniques in vivo using non-invasive bioluminescent imaging (BLI) methods. Results Plasmid DNA carrying the firefly luciferase (LUC) reporter gene under the control of the human Cytomegalovirus (CMV) promoter/enhancer was transfected into mouse skin and liver using biolistic methods. The plasmids were coupled to gold microspheres (1 μm diameter) using different DNA Loading Ratios (DLRs), and "shot" into target tissues using a helium-driven gene gun. The optimal DLR was found to be in the range of 4-10. Bioluminescence was measured using an In Vivo Imaging System (IVIS-50) at various time-points following transfer. Biolistic gene transfer to mouse skin produced peak reporter gene expression one day after transfer. Expression remained detectable through four days, but declined to undetectable levels by six days following gene transfer. Maximum depth of tissue penetration following biolistic transfer to abdominal skin was 200-300 μm. Similarly, biolistic gene transfer to mouse liver in vivo also produced peak early expression followed by a decline over time. In contrast to skin, however, liver expression of the reporter gene was relatively stable 4-8 days post-biolistic gene transfer, and remained detectable for nearly two weeks. Conclusions The use of bioluminescence imaging techniques enabled efficient evaluation of reporter gene expression in vivo. Our results demonstrate that different tissues show different expression kinetics following gene transfer of the same reporter plasmid to different mouse tissues in vivo. We evaluated superficial (skin) and abdominal organ (liver) targets, and found that reporter gene expression peaked within the first two days post-transfer in each case, but declined most rapidly in the skin (3-4 days) compared to liver (10-14 days). This information is essential for designing effective gene therapy strategies in different target tissues.
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Affiliation(s)
- Jixiang Xia
- Burnett School of Biomedical Sciences, University of Central Florida College of Medicine, Orlando, FL 32827, USA
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Tavernier G, Andries O, Demeester J, Sanders NN, De Smedt SC, Rejman J. mRNA as gene therapeutic: How to control protein expression. J Control Release 2011; 150:238-47. [DOI: 10.1016/j.jconrel.2010.10.020] [Citation(s) in RCA: 150] [Impact Index Per Article: 10.7] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/20/2010] [Accepted: 10/13/2010] [Indexed: 10/18/2022]
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Díez S, Navarro G, de ILarduya CT. In vivo targeted gene delivery by cationic nanoparticles for treatment of hepatocellular carcinoma. J Gene Med 2009; 11:38-45. [PMID: 19021130 DOI: 10.1002/jgm.1273] [Citation(s) in RCA: 58] [Impact Index Per Article: 3.6] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/09/2023] Open
Abstract
BACKGROUND Transgene expression in vivo for therapeutic purposes will require methods that allow for efficient gene transfer into cells. Although current vector technologies are being improved, the development of novel vector systems with improved targeting specificity, higher transduction efficiencies and improved safety is necessary. METHODS Asialoglycoprotein receptor-targeted cationic nanoparticles for interleukin (IL)-12 encapsulation (NP1) or adsorption (NP2) have been formulated by blending poly(D,L-lactic-co-glycolic) acid (PLGA) (50 : 50) with the cationic lipid 1,2-dioleoyl-3-(trimethylammonium) propane (DOTAP) and the ligand asialofetuin (AF), by using a modified solvent evaporation process. RESULTS We present a novel targeted lipopolymeric vector, which improves significantly the levels of luciferase gene expression in the liver upon i.v. administration. Targeted-NP2 particles showed a five- and 12-fold higher transfection activity in the liver compared to non-targeted (plain) complexes or naked pCMV DNA, respectively. On the other hand, BNL tumor-bearing animals treated with AF-NP1 containing the therapeutic gene IL-12, showed tumor growth inhibition, leading to a complete tumor regression in 75% of the treated mice, without signs of recurrence. High levels of IL-12 and interferon-gamma were detected in the sera of treated animals. Mice survival also improved considerably. Tumor treatment with AF-NP2 formulations lead only to a retardation in the tumor growth. CONCLUSIONS In the present study, we have developed an efficient targeted non-viral vector for IL-12 gene transfer in hepatocellular carcinoma in vivo, by employing non-toxic cationic PLGA/DOTAP/AF nanoparticles. These results demonstrate for the first time that this cationic system could be used successfully and safely for delivery of therapeutic genes with antitumor activity into liver tumors with targeting specificity.
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Affiliation(s)
- Sonsoles Díez
- Department of Pharmacy and Pharmaceutical Technology, School of Pharmacy, University of Navarra, Pamplona, Spain
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11
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Yeikilis R, Gal S, Kopeiko N, Paizi M, Pines M, Braet F, Spira G. Hydrodynamics based transfection in normal and fibrotic rats. World J Gastroenterol 2006; 12:6149-55. [PMID: 17036386 PMCID: PMC4088108 DOI: 10.3748/wjg.v12.i38.6149] [Citation(s) in RCA: 20] [Impact Index Per Article: 1.1] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 02/07/2023] Open
Abstract
AIM: Hydrodynamics based transfection (HBT), the injection of a large volume of naked plasmid DNA in a short time is a relatively simple, efficient and safe method for in vivo transfection of liver cells. Though used for quite some time, the mechanism of gene transfection has not yet been elucidated.
METHODS: A luciferase encoding plasmid was injected using the hydrodynamics based procedure into normal and thioacetamide-induced fibrotic Sprague Dawley rats. Scanning and transmission electron microscopy images were taken. The consequence of a dual injection of Ringer solution and luciferase pDNA was followed. Halofuginone, an anti collagen type I inhibitor was used to reduce ECM load in fibrotic rats prior to the hydrodynamic injection.
RESULTS: Large endothelial gaps formed as soon as 10’ following hydrodynamic injection; these gradually returned to normal 10 d post injection. Hydrodynamic administration of Ringer 10 or 30 m prior to moderate injection of plasmid did not result in efficient transfection suggesting that endothelial gaps by themselves are not sufficient for gene expression. Gene transfection following hydrodynamic injection in thioacetamide induced fibrotic rats was diminished coinciding with the level of fibrosis. Halofuginone, a specific collagen typeIinhibitor, alleviated this effect.
CONCLUSION: The hydrodynamic pressure formed following HBT results in the formation of large endothelial gaps. These gaps, though important in the transfer of DNA molecules from the blood to the space of Disse are not enough to provide the appropriate conditions for hepatocyte transfection. Hydrodynamics based injection is applicable in fibrotic rats provided that ECM load is reduced.
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Affiliation(s)
- Rita Yeikilis
- Department of Anatomy and Cell Biology, Faculty of Medicine, POB 9649, Haifa 31096, Israel
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Uchida M, Natsume H, Kishino T, Seki T, Ogihara M, Juni K, Kimura M, Morimoto Y. Immunization by particle bombardment of antigen-loaded poly-(DL-lactide-co-glycolide) microspheres in mice. Vaccine 2005; 24:2120-30. [PMID: 16356602 DOI: 10.1016/j.vaccine.2005.11.027] [Citation(s) in RCA: 11] [Impact Index Per Article: 0.6] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/31/2005] [Revised: 10/20/2005] [Accepted: 11/07/2005] [Indexed: 11/27/2022]
Abstract
In the present study, we investigated whether poly-(DL-lactide-co-glycolide) (50:50) microspheres (PLG MS) containing a model antigen, ovalbumin (OVA), were delivered into mouse skin and the immune responses induced using a microparticulate bombardment system, Helios gene gun system, which can painlessly deliver the powdered drug through the stratum corneum to the epidermal-dermal interface using a high velocity supersonic flow of helium gas to accelerate the particles. The introduction of OVA-loaded PLG MS shows helium pressure-dependence, so that improved introduction can be achieved by a higher helium pressure used, thereby inducing sufficient anti-OVA IgG level. Moreover, in order to determine the type of immune system induced using particle bombardment, we investigated helper T-cell response characterized by the cytokine production in the isolated splenocytes 6 weeks after immunization and consequent production of the anti-OVA IgG subclasses in the serum in mice. As a result, IL-4 production in splenocytes and anti-OVA IgG1 level were preferentially elicited by particle bombardment with OVA-loaded PLG MS compared with IFN-gamma and anti-OVA IgG2a level. It seemed likely that particle bombardment using this system led to a Th-2 type immune response, i.e. a humoral immune response. In conclusion, this microparticulate bombardment system is a promising immunization method, expected to become an alternative to needle injection used to administer a broad range of vaccines for the treatment of various diseases.
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Affiliation(s)
- Masaki Uchida
- Faculty of Pharmaceutical Sciences, Josai University, Sakado, Saitama 350-0295, Japan
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Liu F, Tyagi P. Naked DNA for Liver Gene Transfer. ADVANCES IN GENETICS 2005; 54:43-64. [PMID: 16096007 DOI: 10.1016/s0065-2660(05)54003-3] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 05/03/2023]
Abstract
The majority of acquired and inherited genetic disorders, including most inborn errors of metabolism, are manifested in the liver. Therefore, it is hardly any surprise to see a large number of Medline reports describing gene therapy efforts in preclinical settings directed toward this organ (Inoue et al., 2004; Oka and Chen, 2004). Of late, non-viral vectors have garnered a lot of attention from the biomedical research community engaged in liver gene therapy (Gupta et al., 2004). However, the first initiative toward gene transfer to the liver using a non-viral approach was taken by Hickman et al. (1994), who applied the technique of naked DNA injection pioneered by Wolff (1990) for skeletal muscle. Direct injection of naked DNA resulted in low, variable and localized gene expression in the rat liver. Consequently, several developments reported in the literature since then aimed to improve hepatic gene expression by employing both surgical and nonsurgical methods. These developments include the exploitation of the unique vasculature of liver as well as the use of electric and mechanical force as an adjunct to the systemic administration of the naked plasmid gene. This chapter focuses on these developments reported from various laboratories, including ours. In addition, the underlying mechanism responsible for the dramatic increase in gene expression using these latest approaches for non-viral gene transfer to the liver is also discussed.
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Affiliation(s)
- Feng Liu
- Center for Pharmacogenetics, School of Pharmacy University of Pittsburgh, Pittsburgh, Pennsylvania 15261, USA
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Sakai M, Nishikawa M, Thanaketpaisarn O, Yamashita F, Hashida M. Hepatocyte-targeted gene transfer by combination of vascularly delivered plasmid DNA and in vivo electroporation. Gene Ther 2004; 12:607-16. [PMID: 15616599 DOI: 10.1038/sj.gt.3302435] [Citation(s) in RCA: 40] [Impact Index Per Article: 1.9] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/09/2022]
Abstract
To increase transgene expression in the liver, electric pulses were applied to the left lateral lobe after intravenous injection of naked plasmid DNA (pDNA) or pDNA/liver targeting vector complex prepared with galactosylated poly(L-lysine) or galactosylated polyethyleneimine. Electroporation (250 V/cm, 5 ms/pulse, 12 pulses, 4 Hz) after naked pDNA injection dramatically increased the expression up to 200,000-fold; the expression level obtained was significantly greater than that achieved by the combination of pDNA/vector complex and electroporation. We clearly demonstrated that the expression was dependent on the plasma concentration of pDNA at the time when the electric pulses were applied. Separation of liver cells revealed that the distribution of naked pDNA as well as transgene expression was largely selective to hepatocytes in the electroporated lobe. The number of cells expressing transgene product using vascularly administered naked pDNA followed by electroporation was significantly (P<0.01) greater and more widespread than that obtained by local injection of naked pDNA. These results indicate that the application of in vivo electroporation to vascularly administered naked pDNA is a useful gene transfer approach to a large number of hepatocytes.
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Affiliation(s)
- M Sakai
- Department of Drug Delivery Research, Graduate School of Pharmaceutical Sciences, Kyoto University, Sakyo-ku, Kyoto 606-8501, Japan
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Liu F, Lei J, Vollmer R, Huang L. Mechanism of liver gene transfer by mechanical massage. Mol Ther 2004; 9:452-7. [PMID: 15006613 DOI: 10.1016/j.ymthe.2003.12.003] [Citation(s) in RCA: 33] [Impact Index Per Article: 1.6] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/06/2003] [Accepted: 12/08/2003] [Indexed: 11/27/2022] Open
Abstract
Many metabolic diseases are caused by defects in the metabolic pathways in the liver. Others result from the absence of specific proteins normally produced and secreted by the liver. Because these metabolic disorders are usually caused by single gene defect, they are ideal candidates for gene therapy. We have previously shown that mouse liver can be transfected by mechanically massaging the liver (MML) after intravenous injection of naked plasmid DNA. We now show a significant linear relationship between the level of liver gene expression and the venous blood pressure, supporting the idea that gene transfer by MML is due, at least in part, to pressure-mediated effect. Liver transfection could not be blocked by co-injection of excess irrelevant DNA or poly I, suggesting that there is no involvement of receptors, including the scavenger receptor, in MML. Moreover, the level of gene expression could be further enhanced by a combination of MML and an increase in DNA retention-time in the liver. Persistence of gene expression could be significantly improved using an EBV-based plasmid vector. Our data suggest the mechanical massage produces transient membrane defects through which naked DNA can enter into the liver cells by simple diffusion.
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Affiliation(s)
- Feng Liu
- School of Pharmacy, University of Pittsburgh, Pittsburgh, PA 15213, USA.
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Matsuno Y, Iwata H, Umeda Y, Takagi H, Mori Y, Miyazaki JI, Kosugi A, Hirose H. Nonviral gene gun mediated transfer into the beating heart. ASAIO J 2004; 49:641-4. [PMID: 14655727 DOI: 10.1097/01.mat.0000093746.63497.ae] [Citation(s) in RCA: 24] [Impact Index Per Article: 1.1] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/26/2022] Open
Abstract
Several techniques for gene transfer into the heart have been developed, including direct injection of naked plasmid DNA into the myocardium and coronary infusion of various viral vectors. However, complications and side effects with those methods have been reported. In this study, to resolve these problems, the authors investigated the feasibility of nonviral gene transfer into the beating heart with the hand held gene gun. The genes pCAGGS/CTLA4-EGFP were coated around the surface of gold particles. Three sizes of gold particles (0.6, 1.0, and 1.6 microm in diameter) and three settings of helium gas pressure (200, 250, and 300 psi) were examined. Gene transfer into the rat beating heart was performed using the hand held gene gun. EGFP expressions were detected by fluorescence microscopy from day 1 to 3 weeks after bombardment. The most prominent expressions were detected with the combination of 1.0 microm gold particles and 300 psi helium gas pressure. In this study, the present authors showed that non-viral gene transfer into the beating heart was feasible with the hand held gene gun. This technique is effective for gene transfer into the heart and may be one of the most useful methods for gene therapy for many cardiovascular diseases in the future.
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Affiliation(s)
- Yukihiro Matsuno
- First Department of Surgery, Gifu University School of Medicine, Gifu, Japan
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Yoshizawa J, Li XK, Fujino M, Kimura H, Mizuno R, Hara A, Ashizuka S, Kanai M, Kuwashima N, Kurobe M, Yamazaki Y. Successful in utero gene transfer using a gene gun in midgestational mouse fetuses. J Pediatr Surg 2004; 39:81-4. [PMID: 14694377 DOI: 10.1016/j.jpedsurg.2003.09.001] [Citation(s) in RCA: 14] [Impact Index Per Article: 0.7] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 10/26/2022]
Abstract
BACKGROUND/PURPOSE In utero gene therapy offers a number of potential advantages over postnatal gene therapy. A latest method of gene transfer to fetuses in utero uses a new tool called a gene gun. The gene gun is less invasive and simpler than other in utero methods. The current study was designed to determine whether the gene gun is an effective tool for transferring genes to mouse fetuses in utero. METHODS Using a gene gun, we transferred plasmids that included enhanced green fluorescent protein (EGFP) genes and cytomegalo virus promoters to the abdominal skin of 40 A/J fetal mice at each of 3 gestational ages (13, 14, or 15 days). Four or 5 days after gene transfer, the number of surviving fetuses was counted, and a color image of EGFP in the skin was analyzed for gene transfer rates by fluorescence microscopy. Survival rates were analyzed using Fisher's Exact test. RESULTS The mean survival rate was 89.2% (107 of 120) in gene transfer fetuses and 91.7% (55 of 60) in controls. There is no difference in survival rate between gene transfer fetus and control. The highest gene transfer rate was 100% (37 of 37) at the gestational age of 14 days. The rate was 97.1% (34 of 35) at gestational ages of 13 and 15 days. CONCLUSIONS The results of this study show that in utero gene transfer by gene gun is a less-invasive technique, and the gene gun is an effective tool transferring genes to mouse fetuses in utero.
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Affiliation(s)
- Jyoji Yoshizawa
- Division of Pediatric Surgery, Department of Surgery, The Jikei University School of Medicine, Tokyo, Japan
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18
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Nakamura M, Wang J, Murakami T, Ajiki T, Hakamata Y, Kaneko T, Takahashi M, Okamoto H, Mayumi M, Kobayashi E. DNA immunization of the grafted liver by particle-mediated gene gun1. Transplantation 2003; 76:1369-75. [PMID: 14627917 DOI: 10.1097/01.tp.0000091118.22413.e1] [Citation(s) in RCA: 8] [Impact Index Per Article: 0.4] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/05/2023]
Abstract
BACKGROUND Direct DNA vaccination of liver allografts before transplantation may provide an effective strategy for inducing protective immunity to infection and malignancy. METHODS In this study, the authors examined the feasibility of gene gun-mediated vaccination of liver grafts. Using plasmids expressing luciferase and green fluorescent proteins, their expression was tested in a graft liver. RESULTS Protein expression was observed in the graft liver and significantly enhanced in hepatectomized rats. A short course of tacrolimus (FK506) also evoked the expression of these proteins. Effects of primary immunization to the liver on the humoral response were then tested using an expression plasmid encoding hepatitis B virus surface (HBs) antigen and were compared to that of skin immunization alone. The results showed that local immunization to the liver strongly induced antibody formation. Furthermore, the combination of an immunized partial liver graft with tacrolimus significantly enhanced antibody production against HBs antigen. CONCLUSIONS A DNA vaccine to the liver may be one strategy for preventing infectious disease associated with liver transplantation under tacrolimus treatment.
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Affiliation(s)
- Masahiko Nakamura
- Division of Organ Replacement Research and Molecular Immunology, Jichi Medical School, Tochigi, Japan
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19
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Wirth MJ, Wahle P. Biolistic transfection of organotypic cultures of rat visual cortex using a handheld device. J Neurosci Methods 2003; 125:45-54. [PMID: 12763229 DOI: 10.1016/s0165-0270(03)00024-4] [Citation(s) in RCA: 19] [Impact Index Per Article: 0.9] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 10/27/2022]
Abstract
Our aim is the biolistic transfection of organotypic cultures of rat visual cortex with plasmids coding for neurotrophic factors, which then become expressed for limited periods of time during postnatal ontogenesis. Out of two commercially available devices, we adopted the handheld 'Helios Gene Gun' instead of the stationary PDS-1000He (both Biorad, Munich, Germany). This device allows multiple transfections of single targets and the transfection of a distinct part of a co-culture when utilising an aperture. Unfortunately, the most detailed protocols are limited to the stationary device and not compatible with the hand-held device. We report here the construction of a support for the gene gun including an aperture and the establishment of a protocol to efficiently transfect rat cortical slice cultures. We achieve a high degree of co-expression of independent plasmids coated on the same particles. The expression of the neurotrophin plasmids is demonstrated on mRNA and protein level.
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Affiliation(s)
- Marcus J Wirth
- AG Entwicklungsneurobiologie, Fakultät für Biologie, Ruhr-Universität, ND 6/56a, D-44780, Bochum, Germany.
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20
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Ajiki T, Murakami T, Kobayashi Y, Hakamata Y, Wang J, Inoue S, Ohtsuki M, Nakagawa H, Kariya Y, Hoshino Y, Kobayashi E. Long-lasting gene expression by particle-mediated intramuscular transfection modified with bupivacaine: combinatorial gene therapy with IL-12 and IL-18 cDNA against rat sarcoma at a distant site. Cancer Gene Ther 2003; 10:318-29. [PMID: 12679805 DOI: 10.1038/sj.cgt.7700575] [Citation(s) in RCA: 18] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/08/2022]
Abstract
The immune response is modulated by genetic adjuvants using plasmid vectors expressing cytokines. Skeletal muscle can express a foreign gene intramuscularly administered via a needle injection, and the potential of muscle as a target tissue for somatic gene therapy in treating cancer has been explored. In the present study, we investigated the efficacy of particle-mediated intramuscular transfection modified with a local anesthetic agent, bupivacaine, on luciferase and green fluorescent protein. The results indicate that these proteins are more efficiently expressed and persist longer in muscle modified in this way compared with the needle-injection method. Using an established rat sarcoma model, particle-mediated intramuscular gene-gun therapy with a combination of IL-12 and IL-18 cDNA was conducted. Growth of the distant sarcoma was significantly inhibited by particle-mediated intramuscular combination gene therapy, and the survival rate was also improved. Furthermore, the combination gene-gun therapy maintained significant levels of interferon-gamma and induced a high activity of tumor-specific cytotoxic T lymphocytes. These results suggest that the sustained local delivery of IL-12 and IL-18 cDNA using intramuscular gene-gun therapy modified with bupivacaine can induce long-term antitumor immunity, and can provide the great advantage of inhibiting the disseminated tumor.
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Affiliation(s)
- Takashi Ajiki
- Division of Organ Replacement Research and Molecular Immunology, Center for Molecular Medicine, Jichi Medical School, 3311-1 Yakushiji, Minamikawachi-machi, Kawachi-gun, Tochigi 329-0498, Japan
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21
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Arangoa MA, Düzgüneş N, Tros de Ilarduya C. Increased receptor-mediated gene delivery to the liver by protamine-enhanced-asialofetuin-lipoplexes. Gene Ther 2003; 10:5-14. [PMID: 12525832 DOI: 10.1038/sj.gt.3301840] [Citation(s) in RCA: 62] [Impact Index Per Article: 2.8] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 02/04/2023]
Abstract
A novel lipidic vector composed of DOTAP/Chol liposomes, asialofetuin (AF), protamine sulfate and DNA has been developed. The resulting protamine-AF-lipoplexes improved significantly the levels of gene expression in cultured cells and in the liver upon i.v. administration. Lipoplexes containing the optimal amount of AF (1 microg/microg DNA) showed a 16-fold higher transfection activity in HepG2 cells than non-targeted (plain) complexes. The uptake by cells having asialoglycoprotein receptors (ASGPr) on their plasma membrane was decreased by the addition of free AF, indicating that AF-lipoplexes were taken up specifically by cells via ASGPr-mediated endocytosis. Results from transfections performed in cells defective in ASGPr, ie HeLa cells, confirmed this mechanism. By addition of the condensing peptide, protamine sulfate, smaller complexes were obtained, which enhanced even more the uptake of AF-complexes in HepG2 cells and in the liver. The optimal amount of protamine was 0.4 microg/mcirog DNA, and gene expression was about 5-fold over that obtained with AF-lipoplexes in the absence of the peptide, and 75-fold higher than that with plain conventional lipoplexes. Protamine-AF-lipoplexes increased by a factor of 12 luciferase gene expression in the liver of mice administered systemically via the tail vein, compared to plain complexes. In summary, our findings extend the scope of previous studies where AF-lipoplexes were used to introduce DNA into hepatocytes. The combination of targeting and protamine condensation obviated the need for partial hepatectomy, commonly required to obtain efficient gene delivery in this organ. Since protamine sulfate has been proven to be non-toxic in humans, the novel liver-specific vector described here may be useful for the delivery of clinically important genes to this organ.
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Affiliation(s)
- M A Arangoa
- Department of Pharmacy and Pharmaceutical Technology, School of Pharmacy, University of Navarra, Pamplona. Spain
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22
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Abstract
With the recent completion of the human genome project and the tremendous growth of biotechnology, the desire to extract information concerning gene expression, protein level, subcellular localization, and functionality in the liver will demand the development of efficient gene transfer to this organ with minimal toxicity. In this report, we show that significant gene expression in the liver could be achieved by simple mechanical massage after intravenous injection of naked plasmid DNA into mice. This method is simple, highly reproducible, repeatable, and, more importantly, free of toxicity. Hepatic gene transfer with hepatocyte growth factor (HGF) plasmid DNA prevented endotoxin-induced lethal fulminant hepatic failure, leading to dramatically enhanced survival in mice.
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Affiliation(s)
- Feng Liu
- Center for Pharmacogenetics, School of Pharmacy, University of Pittsburgh, Pittsburgh, PA 15213, USA.
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23
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O'Brien JA, Holt M, Whiteside G, Lummis SC, Hastings MH. Modifications to the hand-held Gene Gun: improvements for in vitro biolistic transfection of organotypic neuronal tissue. J Neurosci Methods 2001; 112:57-64. [PMID: 11640958 DOI: 10.1016/s0165-0270(01)00457-5] [Citation(s) in RCA: 70] [Impact Index Per Article: 2.9] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/26/2022]
Abstract
Transfection and subsequent expression of DNA in living neuronal tissue is problematic and no technique has emerged that is completely non-damaging, efficient and reproducible. The Bio-Rad hand-held Gene Gun has overcome some of these problems by exploiting a biolistic method in which small gold particles carrying plasmid DNA are propelled into neurons whilst causing minimal detectable cell damage. In its current configuration, however, the Bio-Rad Gene Gun is optimised for transfecting cells in dispersed cultures, and therefore delivers particles superficially over a relatively wide area. Here we report modifications to the Bio-Rad Gene Gun that both enhance its accuracy by restricting its target area, and increase the depth penetration achieved by gold particles, thereby allowing smaller and deeper tissues to be transfected. These alterations make the modified Gene Gun more applicable for in vitro transfection of organotypic cultures and enhance its potential utility for in vivo gene delivery. Moreover, the modified configuration operates successfully at lower gas pressures, thereby reducing even further the degree of cell damage incurred during transfection.
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Affiliation(s)
- J A O'Brien
- Neurobiology Division, MRC Laboratory of Molecular Biology, Hills Road, Cambridge CB2 2QH, UK
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24
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Liu F, Huang L. Improving plasmid DNA-mediated liver gene transfer by prolonging its retention in the hepatic vasculature. J Gene Med 2001; 3:569-76. [PMID: 11778903 DOI: 10.1002/jgm.222] [Citation(s) in RCA: 32] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/08/2022] Open
Abstract
BACKGROUND Naked DNA is the simplest and safest method to deliver genes to the liver. In this study, we demonstrate that significant gene expression could be achieved in the liver by transiently restricting blood flow through the liver immediately following peripheral intravenous injection of plasmid DNA. METHODS Mice were intravenously (tail vein) injected with plasmid DNA in 100 microl of saline (0.9% NaCl) immediately followed by 8 s of occlusion of blood flow through the liver. The occlusion of blood flow was performed by using a clip at either the vena cava (VC) or at the portal vein and hepatic artery (PV+HA). Alternatively, the VC was clamped for 4 s followed by clamping the PV+HA for 4 s (VC and PV+HA). RESULTS Gene transfer to the liver was completed after blood flow through the liver was blocked for as short as 1 s. Up to 560 pg of luciferase protein per mg of extracted protein was observed from the liver after a single injection of 80 microg of plasmid DNA. Gene expression was increased more than 50-fold by the combination of clamping and electroporation. CONCLUSION This is the first demonstration of gene transfer to the liver via systemic administration without using any carrier system or physical force. Also, the technique provides new insights into the mechanism of hepatic gene transfer.
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Affiliation(s)
- F Liu
- Center for Pharmacogenetics, School of Pharmacy, University of Pittsburgh, PA 15213, USA. fliu+@pitt.edu
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25
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Wang J, Murakami T, Hakamata Y, Ajiki T, Jinbu Y, Akasaka Y, Ohtsuki M, Nakagawa H, Kobayashi E. Gene gun-mediated oral mucosal transfer of interleukin 12 cDNA coupled with an irradiated melanoma vaccine in a hamster model: successful treatment of oral melanoma and distant skin lesion. Cancer Gene Ther 2001; 8:705-12. [PMID: 11687893 DOI: 10.1038/sj.cgt.7700363] [Citation(s) in RCA: 31] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/25/2001] [Accepted: 06/11/2000] [Indexed: 11/08/2022]
Abstract
Malignant melanoma involving the oral cavity has a highly metastatic potential. Curative surgery is required to resect extensive oral tissues and often results in dysfunction as well as a severe cosmetic deformity in patients with the disease. An alternative technology for the local and sustained delivery of cytokines for cancer immunotherapy has been shown to induce tumor regression, suppression of metastasis, and development of systemic antitumor immunity. However, local immunization of the oral cavity has not previously been studied. In this study, we examined the efficacy of particle-mediated oral gene transfer on luciferase and green fluorescent protein production. The results showed that these proteins were more significantly expressed in oral mucosa than the skin, stomach, liver, and muscle. Using an established oral melanoma model in hamsters, particle-mediated oral gene gun therapy with interleukin (IL) 12 cDNA was then conducted. The results indicated that direct bombardment of mouse IL-12 cDNA suppressed tumor formation and improved the survival rate. The skin tumor model created by inoculation of melanoma cells was also significantly inhibited by the oral bombardment of IL-12 cDNA coupled with an irradiated melanoma vaccine administrated to the oral mucosa, compared to treatment with a percutaneous vaccine. IL-12 gene gun therapy, combined with an oral mucosal vaccine, induced interferon-gamma mRNA expression in the host spleen for a long time. These results suggest that immunization of oral mucosa may induce systemic antitumor immunity more efficiently than immunization of the skin and that oral mucosa may be one of the most suitable tissues for cancer gene therapy by means of particle-mediated gene transfer.
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Affiliation(s)
- J Wang
- Division of Organ Replacement Research, Center for Molecular Medicine, Jichi Medical School, Tochigi 329-0498, Japan
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26
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Sohn RL, Murray MT, Schwarz K, Nyitray J, Purray P, Franko AP, Palmer KC, Diebel LN, Dulchavsky SA. In-vivo particle mediated delivery of mRNA to mammalian tissues: ballistic and biologic effects. Wound Repair Regen 2001; 9:287-96. [PMID: 11679137 DOI: 10.1046/j.1524-475x.2001.00287.x] [Citation(s) in RCA: 20] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/20/2022]
Abstract
Biolistic transmission of mRNA provides transient gene therapy to in vivo organs. This study documents particle mediated mRNA transmission to a solid organ and wound healing model using the mRNA of Green Fluorescent Protein to determine optimal delivery parameters. Renal function, bullet penetration, cellular injury, and Green Fluorescent Protein synthesis were quantified. Chimeric human epidermal growth factor-FLAG epitope cDNA or mRNA was transmitted to wounds in normal or steroid treated animals. Wound bursting strength, human epidermal growth factor-FLAG, and collagen synthesis were determined. Injury and bullet penetration correlated with the delivery velocity and bullet size. Optimal delivery parameters were established which provided widespread Green Fluorescent Protein synthesis. Human epidermal growth factor-FLAG treatment significantly increased collagen content and wound breaking strength in normal and steroid treated animals. FLAG protein synthesis was evident in mRNA treated fascia following treatment. We found the gene gun provides a novel method for efficient, in vivo delivery of mRNA-based therapeutic strategies to mammalian organs with minimal histologic damage allowing transient expression of protein in in vivo target tissues. Co-delivery of Green Fluorescent Protein mRNA may provide a useful positive control to determine effective transmission. Biolistic transmission of human epidermal growth factor-FLAG mRNA provides increased tissue epidermal growth factor levels and accelerates wound healing in normal and steroid exposed animals.
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Affiliation(s)
- R L Sohn
- Department of Surgery, Wayne State University School of Medicine, Detroit, Michigan, USA
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27
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Neuronal apoptosis by apolipoprotein E4 through low-density lipoprotein receptor-related protein and heterotrimeric GTPases. J Neurosci 2001. [PMID: 11069947 DOI: 10.1523/jneurosci.20-22-08401.2000] [Citation(s) in RCA: 62] [Impact Index Per Article: 2.6] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/21/2022] Open
Abstract
The epsilon4 genotype of apolipoprotein E (apoE4) is the most established predisposing factor in Alzheimer's disease (AD); however, it remains unclear how apoE4 contributes to the pathophysiology. Here, we report that the apoE4 protein (ApoE4) evokes apoptosis in neuronal cells through the low-density lipoprotein receptor-related protein (LRP) and heterotrimeric GTPases. We examined neuron/neuroblastoma hybrid F11 cells and found that these cells were killed by 30 microg/ml ApoE4, but not by 30 microg/ml ApoE3. ApoE4-induced death occurred with typical features for apoptosis in time- and dose-dependent manners, and was observed in SH-SY5Y neuroblastomas, but not in glioblastomas or non-neuronal Chinese hamster ovary cells. Activated, but not native, alpha2-macroglobulin suppressed this ApoE4 toxicity. Suppression by the antisense oligonucleotide to LRP and inhibition by low nanomolar concentrations of LRP-associated protein RAP provided evidence for the involvement of LRP. The involvement of heterotrimeric GTPases was demonstrated by the findings that (1) ApoE4-induced death was suppressed by pertussis toxin (PTX), but not by heat-inactivated PTX; and (2) transfection with PTX-resistant mutant cDNAs of Galpha(i) restored the toxicity of ApoE4 restricted by PTX. We thus conclude that one of the neurotoxic mechanisms triggered by ApoE4 is to activate a cell type-specific apoptogenic program involving LRP and the G(i) class of GTPases and that the apoE4 gene may play a direct role in the pathogenesis of AD and other forms of dementia.
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28
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Shinoda Y, Suzuki T, Sugawara-Yokoo M, Nagamatsu S, Kuwano H, Takata K. Expression of Sugar Transporters by In Vivo Electroporation and Particle Gun Methods in the Rat Liver: Localization to Specific Membrane Domains. Acta Histochem Cytochem 2001. [DOI: 10.1267/ahc.34.15] [Citation(s) in RCA: 5] [Impact Index Per Article: 0.2] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/09/2023] Open
Affiliation(s)
- Yasuo Shinoda
- First Department of Surgery, Gunma University School of Medicine
- Laboratory of Molecular and Cellular Morphology, Department of Cell Biology, Institute for Molecular and Cellular Regulation, Gunma University
| | - Takeshi Suzuki
- Laboratory of Molecular and Cellular Morphology, Department of Cell Biology, Institute for Molecular and Cellular Regulation, Gunma University
| | - Minako Sugawara-Yokoo
- Laboratory of Molecular and Cellular Morphology, Department of Cell Biology, Institute for Molecular and Cellular Regulation, Gunma University
- Third Department of Internal Medicine, Gunma University School of Medicine
| | - Shinya Nagamatsu
- Department of Biochemistry, Kyorin University School of Medicine,
| | - Hiroyuki Kuwano
- First Department of Surgery, Gunma University School of Medicine
| | - Kuniaki Takata
- Laboratory of Molecular and Cellular Morphology, Department of Cell Biology, Institute for Molecular and Cellular Regulation, Gunma University
- Department of Anatomy and Cell Biology, Gunma University School of Medicine
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29
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Yoshida S, Kashiwamura SI, Hosoya Y, Luo E, Matsuoka H, Ishii A, Fujimura A, Kobayashi E. Direct immunization of malaria DNA vaccine into the liver by gene gun protects against lethal challenge of Plasmodium berghei sporozoite. Biochem Biophys Res Commun 2000; 271:107-15. [PMID: 10777689 DOI: 10.1006/bbrc.2000.2558] [Citation(s) in RCA: 28] [Impact Index Per Article: 1.1] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/22/2022]
Abstract
The liver is the first target organ for malaria parasites immediately after the bite of an infected mosquito. We studied local immunization of malaria DNA vaccines at the site of the liver using a gene gun as a useful tool for in vivo transfection of foreign genes. A malaria DNA vaccine consisting of the Plasmodium berghei circumsporozoite protein (PbCSP) gene plus the mouse IL-12 gene was bombarded directly by a gene gun into mouse liver once or into the skin twice. A marked protective effect was induced by gene bombardment into the liver (more than 71%) compared with that into the skin (less than 33%). A Th1-type immune response and high production of iNOS were observed in the hepatic lymphocytes from mice bombarded into the liver, resulting in more effective protection compared with those bombarded into the skin. These results provide an important implication on the development of efficient malaria vaccine strategies.
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Affiliation(s)
- S Yoshida
- Department of Medical Zoology, Center for Molecular Medicine, Jichi Medical School, 3311-1 Yakushiji, Minamikawachimachi, Tochigi, Japan.
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30
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Sato H, Hattori S, Kawamoto S, Kudoh I, Hayashi A, Yamamoto I, Yoshinari M, Minami M, Kanno H. In vivo gene gun-mediated DNA delivery into rodent brain tissue. Biochem Biophys Res Commun 2000; 270:163-70. [PMID: 10733922 DOI: 10.1006/bbrc.2000.2369] [Citation(s) in RCA: 28] [Impact Index Per Article: 1.1] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/22/2022]
Abstract
Various types of gene transfer into live tissues have been tried. However, in vivo gene transfer into brain tissue or neuronal cells without virus vector has required a great effort. Particle-mediated gene transfer into live brain tissue was thought to be impossible because of its fragility and the mechanical problem of a previous type of gene gun. In addition, particle-mediated DNA transfer into monolayer-cultured cells without mechanical damage has been difficult. We successfully transferred DNA into rodent live brain tissue and also into monolayer-cultured cells without mechanical damage by using a new type of gene gun and also confirmed gene expression in the brain. This new method represents another variation of gene transfer into the brain.
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Affiliation(s)
- H Sato
- Department of Neurosurgery, Yokohama City University School of Medicine, Fukuura 3-9, Kanazawa-ku, Yokohama, 236-0004, Japan
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31
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Qian C, Drozdzik M, Caselmann WH, Prieto J. The potential of gene therapy in the treatment of hepatocellular carcinoma. J Hepatol 2000; 32:344-51. [PMID: 10707877 DOI: 10.1016/s0168-8278(00)80082-3] [Citation(s) in RCA: 36] [Impact Index Per Article: 1.4] [Reference Citation Analysis] [MESH Headings] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 01/24/2023]
Affiliation(s)
- C Qian
- Department of Medicine, School of Medicine and Clinica Universitaria, University of Navarra, Pamplona, Spain
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32
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Eizema K, van Heugten HA, Bezstarosti K, van Setten MC, Lamers JM. Endothelin-1 responsiveness of a 1.4 kb phospholamban promoter fragment in rat cardiomyocytes transfected by the gene gun. J Mol Cell Cardiol 2000; 32:311-21. [PMID: 10722806 DOI: 10.1006/jmcc.1999.1076] [Citation(s) in RCA: 6] [Impact Index Per Article: 0.2] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 11/22/2022]
Abstract
The transcriptional regulation of an isolated rat phospholamban (PL) promoter fragment in rat cardiomyocytes was analyzed by applying a new method to reach substantially higher transfection efficiencies: gene gun biolistics. The gene gun transfection method was optimized for application to primary cultures of rat neonatal cardiomyocytes. Cells, cultured at different densities (0.75-1.50x10(5)cells/cm(2)) in serum-free medium, were transfected with DNA coated gold particles. A transfection efficiency of up to 10% could be achieved (compared to <1% with other methods) by the gene gun as checked using a RSV- beta-Gal construct. Cardiomyocytes were stimulated by endothelin-1 (ET-1) (10(-8)M) to induce hypertrophy, thereby yielding the characteristic changes in gene expression (upregulation of Atrial Natriuretic Factor (ANF) and downregulation of PL). The basal activity of an ANF promoter fragment (increasing from the lowest to highest density 2.6-fold) and its ET-1 inducibility (only significant upregulation of 2.6-fold, at lowest density) appeared to be dependent on the plating density of the cardiomyocytes. A PL promoter fragment was isolated, sequenced and 1.4 kb was subcloned in a luciferase reporter vector. The basal activity of the PL promoter fragment was not dependent on the plating density. ET-1 did not downregulate the PL promoter, rather a significant upregulation (1.4-fold) was found at the highest plating density. In conclusion, plating density of the cardiomyocytes can influence promoter activity as shown with an ANF promoter fragment. A newly isolated and sequenced rat PL promoter fragment did not direct gene expression as expected on basis of downregulation of the PL gene by ET-1 observed in this model.
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Affiliation(s)
- K Eizema
- Department of Biochemistry, Erasmus University Rotterdam, Rotterdam, 3000 DR, Netherlands
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33
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SERCA2 and ANF Promoter-Activity Studies in Hypertrophic Cardiomyocytes Using Liposome-, Gene Gun-, and Adenovirus-Mediated Gene Transfer. ACTA ACUST UNITED AC 2000. [DOI: 10.1007/978-1-4615-4423-4_5] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.0] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register]
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34
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Yanai K, Hirota K, Taniguchi-Yanai K, Shigematsu Y, Shimamoto Y, Saito T, Chowdhury S, Takiguchi M, Arakawa M, Nibu Y, Sugiyama F, Yagami K, Fukamizu A. Regulated expression of human angiotensinogen gene by hepatocyte nuclear factor 4 and chicken ovalbumin upstream promoter-transcription factor. J Biol Chem 1999; 274:34605-12. [PMID: 10574924 DOI: 10.1074/jbc.274.49.34605] [Citation(s) in RCA: 32] [Impact Index Per Article: 1.2] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/06/2022] Open
Abstract
We previously identified various upstream and downstream regulatory elements and factors important for hepatic expression of the human angiotensinogen (ANG) gene, the precursor of vasoactive octapeptide angiotensin II. In the present study, to further investigate the molecular mechanism of human ANG transcriptional regulation, we generated transgenic mice carrying the fusion gene composed of the 1. 3-kilobase promoter of the human ANG gene, its downstream enhancer, and the chloramphenicol acetyltransferase reporter gene. Because expression of the chloramphenicol acetyltransferase gene was observed strongly in the liver and weakly in the kidney, we suspected that hepatocyte nuclear factor (HNF) 4 with a tissue expression pattern similar to that of the reporter gene would regulate ANG transcription. In vitro assays indicated that HNF4 bound to the promoter elements and strongly activated the ANG transcription, but that chicken ovalbumin upstream promoter transcription factor (COUP-TF), a transcriptional repressor, dramatically repressed human ANG transcription through the promoter elements and the downstream enhancer core elements. Furthermore, COUP-TF dramatically decreased the human ANG transcription in the mouse liver by the Helios Gene Gun system in vivo. These results suggest that an interplay between HNF4 and COUP-TF could be important in hepatic human ANG transcription.
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Affiliation(s)
- K Yanai
- Center for Tsukuba Advanced Research Alliance, University of Tsukuba, Ibaraki 305-8577, Japan
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Wellmann H, Kaltschmidt B, Kaltschmidt C. Optimized protocol for biolistic transfection of brain slices and dissociated cultured neurons with a hand-held gene gun. J Neurosci Methods 1999; 92:55-64. [PMID: 10595703 DOI: 10.1016/s0165-0270(99)00094-1] [Citation(s) in RCA: 49] [Impact Index Per Article: 1.9] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/08/2023]
Abstract
DNA-transfer into postmitotic neurons or neuronal tissues has been a major problem in neurobiology. For this aim different methods have been used such as viral infection, microinjection, lipofection or calcium phosphate precipitation. However, using these techniques, very poor transfection efficiency was achieved except for virus-mediated gene transfer. Though viral infections are very efficient, this method is expensive and labor-intensive, especially when recombination is used to prepare viral vectors. Biolistic gene transfer of neurons represents another promising transfection technique. This technique was originally used to transfect plant cells and has been further developed for gene transfer into neurons or neuronal tissues. Up to now, only a few reports are available where successful biolistic gene transfer into neurons or neuronal tissues could be shown. Transfection efficiencies were only about 2%. Most of the previously published experiments were carried out under vacuum conditions using in-chamber gene gun types. Here we describe an improved method for efficient neuronal cell transfection using a hand-held gene gun. Expression vectors could be successfully transferred into dissociated cultured hippocampal neurons, PC12 cells, cultured cerebellar granule cells and cerebellar brain slices. In cerebellar granule cells and hippocampal neurons, transfection efficiencies of about 10% were reached.
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Affiliation(s)
- H Wellmann
- Molecular Neurobiology Laboratory, Institute of Anatomy, Albert-Ludwigs-University, Freiburg, Germany
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Eizema K, Van Heugten HA, Bezstarosti K, Van Setten MC, Lamers JM. In vitro analysis of SERCA2 gene regulation in hypertrophic cardiomyocytes and increasing transfection efficiency by gene-gun biolistics. Ann N Y Acad Sci 1999; 874:111-24. [PMID: 10415525 DOI: 10.1111/j.1749-6632.1999.tb09229.x] [Citation(s) in RCA: 4] [Impact Index Per Article: 0.2] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/19/2023]
Abstract
The transcriptional downregulation of the SERCA2 gene is studied using neonatal rat cardiomyocytes stimulated with endothelin-1 to induce hypertrophy. Liposome-based transfection of cells with a 1.9 kb SERCA2 promoter fragment directed expression of a reporter gene identical to the downregulation of genomic SERCA2 expression by endothelin-1. Results of a new gene gun technology for transient transfection of cardiomyocytes with a RSV-beta-galactosidase construct are reported. This new method for propelling DNA-coated gold beads into cardiomyocytes is extremely suitable for directly testing promoter/reporter gene DNA constructs since the transfection efficiency (approximately 10%) appears to be higher than traditional transfection methods.
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Affiliation(s)
- K Eizema
- Department of Biochemistry, Erasmus University Rotterdam, Netherlands
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Kibbe M, Billiar T, Tzeng E. Gene therapy and vascular disease. ADVANCES IN PHARMACOLOGY 1999; 46:85-150. [PMID: 10332502 DOI: 10.1016/s1054-3589(08)60470-6] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 02/23/2023]
Affiliation(s)
- M Kibbe
- Department of General Surgery, University of Pittsburgh, Pennsylvania 15261, USA
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Hosoya Y, Kobayashi E, Yoshida Y, Tomizawa N, Fujimura A, Kagawa Y, Kanazawa K. Introduction of cytokine genes into rat solid neoplasms with a hand-held gene gun. ACTA ACUST UNITED AC 1998. [DOI: 10.4993/acrt1992.7.39] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.1] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/21/2022]
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