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Hydrodynamics-based transfection of the liver: entrance into hepatocytes of DNA that causes expression, takes place very early after injection
NAKED DNA-BASED GENE TRANSFER I acetate during a wash step allowed omission of RNA pretreatment. Material generated by this one column procedure was sufficiently pure to allow transfection of cell lines.
139. Optimization of Electroporation Parameters for the Intramuscular Delivery of Plasmids in Pigs Amir S. Khan,1 Louis C. Smith,1 Ronald V. Abruzzese,1 Kathleen K. Cummings,1 Melissa A. Pope,1 Patricia A. Brown,1 Ruxandra Draghia-Akli.1,2 1 Research, Advisys, Inc., The Woodlands, TX; 2Molecular and Cellular Biology, Baylor College of Medicine, Houston, TX. Increased transgene expression after plasmid transfer to the skeletal muscle is obtained with electroporation in many species, but optimum electroporation conditions are not well defined. Using a plasmid with a muscle-specific secreted alkaline phosphatase (SEAP) gene, we have optimized the electroporation conditions in a large mammal (pig). Parameters tested included electric field intensity, number of pulses, lag time between plasmid injection and electroporation, and plasmid delivery volume. The electrode array has a central port through which the injection needle can be inserted into the muscle, such that the plasmid is delivered within the area that is delineated by the surrounding five electrodes. The sequence of events for plasmid administration/electroporation: a) insert electroporation device with the 5-electrode array into the muscle; b) introduce an injection needle through central port, and inject plasmid into the muscle; c) lag time; d) electroporation. The optimum conditions for SEAP expression were electric pulses, between 0.4 and 0.6 Amp constant current, applied 80 sec after the injection of 0.5 mg SEAP-expressing plasmid in a total volume of 2 mL. When a non-delineated site is electroporated, SEAP expression decreased by 74% (45.6 ± 16 pg/ml/kg in the intact electroporation sequence versus 12 ± 2.5 pg/ml/kg in the interrupted sequence at Day 10, ∗(p<0.02). These results demonstrate that electroporation parameters such as amperage, lag time, and the number of pulses are able to regulate the levels of reporter gene expression in pigs.
140. Hydrodynamics-Based Transfection of the Liver: Entrance into Hepatocytes of DNA That Causes Expression, Takes Place Very Early after Injection Fanjam Andrianaivo, Michèle Lecocq, Simone Wattiaux-De Coninck, Robert Wattiaux, Michel Jadot. 1 Chimie Physiologique, Unité de recherche en Physiologie Moléculaire, Facultés Universitaires N-D de la Paix, Namur, Belgium. The mechanism of gene transfer into hepatocytes by the hydrodynamics-based transfection procedure is not clearly understood. It has been shown that after an 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 S54
be responsible of the efficiency of the transfection (Lecocq M et al, J of Gene Med, 5, 142-156, 2003). We have investigated the problem by giving to the mice an hydrodynamic injection of isotonic NaCl, followed at different time intervals by a conventional injection of DNA, cold or labelled with 35S, with cDNA of luciferase as a reporter gene. Then, we have 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 an hydrodynamic injection on the one hand and the expression and DNA uptake and fate on the other hand.. When DNA is given to mice by a conventional injection a few sec 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 35S-DNA, the uptake and the intracellular fate of the bulk of DNA are similar whatever DNA is administered by a single hydrodynamic injection or by a conventional injection given up to at least two hours after an hydrodynamic injection of isotonic NaCl. Our results show that the efficiency of hydrodynamic based transfection depends on a process that takes place very quickly after injection and is not linked to a delay of DNA degradation. They strongly suggest that expression after an 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.
141. Development of a One-Step Column Purification of Minicircle Vector DNA Devoid of Bacterial Sequences That Results in High Level Persistent Transgene Expression In Vivo Zhi-Ying Chen,1 Cheng-Yi He,1 Mark A. Kay.1 Pediatrics & Genetics, Stanford University School of Medicine, Stanford, CA.
1
We have shown that episomal circular DNA vectors free of plasmid bacterial DNA sequences are capable of indefinite high level transgene expression in vivo. The minicircle vector is generated in bacteria from a parental plasmid containing an inducible phiC31 integrase gene, and att P and att B recognition sites flanking the therapeutic expression cassette. Recombination results in the formation of two circular DNA molecules, one containing the plasmid bacterial backbone and the other the eukaryotic expression cassette. Previously, the minicircle was purified away from the plasmid bacterial DNA by cesium chloride banding in an ultracentrifuge (Chen et al., Mol Ther. 8:495, 2003). We have now devised a onestep purification method that results in a high yield of minicircle vector. To do this, two copies of the phiC31 integrase gene under the control of an inducible promoter (BAD), together with the transgene expression cassette flanked by the attB and attP sites, were built into a minicircle-producing plasmid, p2phiC31. An intron encoded restriction enzyme I-Sce I gene, also under the control of the BAD promoter, together with an I-Sce I restriction site, were also built into p2phiC31. Top 10 E. coli was used to amplify p2phiC31 using standard plasmid preparation. To induce the phiC31 integrase-mediated minicircle formation, an over night culture was re-suspended 4 to 1 (vol/vol) in fresh LB broth containing the pBAD inducer 1% L-arabinose and incubated at 32°C with constant shaking for one hour. The temperature of the incubation was increased to 37°C, to optimize I-Sce I enzymatic activity, for an additional 2 hours. Linearization of the plasmid bacterial DNA circle by the restriction enzyme resulted in its rapid degradation by exonucleases in bacteria. The mini-circle expression cassette becomes the only episomal circular DNA and is easily isolated by an affinity purification Molecular Therapy Volume 9, Supplement 1, May 2004
Copyright The American Society of Gene Therapy
139. Optimization of Electroporation Parameters for the Intramuscular Delivery of Plasmids in Pigs Amir S. Khan,1 Louis C. Smith,1 Ronald V. Abruzzese,1 Kathleen K. Cummings,1 Melissa A. Pope,1 Patricia A. Brown,1 Ruxandra Draghia-Akli.1,2 1 Research, Advisys, Inc., The Woodlands, TX; 2Molecular and Cellular Biology, Baylor College of Medicine, Houston, TX. Increased transgene expression after plasmid transfer to the skeletal muscle is obtained with electroporation in many species, but optimum electroporation conditions are not well defined. Using a plasmid with a muscle-specific secreted alkaline phosphatase (SEAP) gene, we have optimized the electroporation conditions in a large mammal (pig). Parameters tested included electric field intensity, number of pulses, lag time between plasmid injection and electroporation, and plasmid delivery volume. The electrode array has a central port through which the injection needle can be inserted into the muscle, such that the plasmid is delivered within the area that is delineated by the surrounding five electrodes. The sequence of events for plasmid administration/electroporation: a) insert electroporation device with the 5-electrode array into the muscle; b) introduce an injection needle through central port, and inject plasmid into the muscle; c) lag time; d) electroporation. The optimum conditions for SEAP expression were electric pulses, between 0.4 and 0.6 Amp constant current, applied 80 sec after the injection of 0.5 mg SEAP-expressing plasmid in a total volume of 2 mL. When a non-delineated site is electroporated, SEAP expression decreased by 74% (45.6 ± 16 pg/ml/kg in the intact electroporation sequence versus 12 ± 2.5 pg/ml/kg in the interrupted sequence at Day 10, ∗(p<0.02). These results demonstrate that electroporation parameters such as amperage, lag time, and the number of pulses are able to regulate the levels of reporter gene expression in pigs.
140. Hydrodynamics-Based Transfection of the Liver: Entrance into Hepatocytes of DNA That Causes Expression, Takes Place Very Early after Injection Fanjam Andrianaivo, Michèle Lecocq, Simone Wattiaux-De Coninck, Robert Wattiaux, Michel Jadot. 1 Chimie Physiologique, Unité de recherche en Physiologie Moléculaire, Facultés Universitaires N-D de la Paix, Namur, Belgium. The mechanism of gene transfer into hepatocytes by the hydrodynamics-based transfection procedure is not clearly understood. It has been shown that after an 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 S54
be responsible of the efficiency of the transfection (Lecocq M et al, J of Gene Med, 5, 142-156, 2003). We have investigated the problem by giving to the mice an hydrodynamic injection of isotonic NaCl, followed at different time intervals by a conventional injection of DNA, cold or labelled with 35S, with cDNA of luciferase as a reporter gene. Then, we have 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 an hydrodynamic injection on the one hand and the expression and DNA uptake and fate on the other hand.. When DNA is given to mice by a conventional injection a few sec 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 35S-DNA, the uptake and the intracellular fate of the bulk of DNA are similar whatever DNA is administered by a single hydrodynamic injection or by a conventional injection given up to at least two hours after an hydrodynamic injection of isotonic NaCl. Our results show that the efficiency of hydrodynamic based transfection depends on a process that takes place very quickly after injection and is not linked to a delay of DNA degradation. They strongly suggest that expression after an 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.
141. Development of a One-Step Column Purification of Minicircle Vector DNA Devoid of Bacterial Sequences That Results in High Level Persistent Transgene Expression In Vivo Zhi-Ying Chen,1 Cheng-Yi He,1 Mark A. Kay.1 Pediatrics & Genetics, Stanford University School of Medicine, Stanford, CA.
1
We have shown that episomal circular DNA vectors free of plasmid bacterial DNA sequences are capable of indefinite high level transgene expression in vivo. The minicircle vector is generated in bacteria from a parental plasmid containing an inducible phiC31 integrase gene, and att P and att B recognition sites flanking the therapeutic expression cassette. Recombination results in the formation of two circular DNA molecules, one containing the plasmid bacterial backbone and the other the eukaryotic expression cassette. Previously, the minicircle was purified away from the plasmid bacterial DNA by cesium chloride banding in an ultracentrifuge (Chen et al., Mol Ther. 8:495, 2003). We have now devised a onestep purification method that results in a high yield of minicircle vector. To do this, two copies of the phiC31 integrase gene under the control of an inducible promoter (BAD), together with the transgene expression cassette flanked by the attB and attP sites, were built into a minicircle-producing plasmid, p2phiC31. An intron encoded restriction enzyme I-Sce I gene, also under the control of the BAD promoter, together with an I-Sce I restriction site, were also built into p2phiC31. Top 10 E. coli was used to amplify p2phiC31 using standard plasmid preparation. To induce the phiC31 integrase-mediated minicircle formation, an over night culture was re-suspended 4 to 1 (vol/vol) in fresh LB broth containing the pBAD inducer 1% L-arabinose and incubated at 32°C with constant shaking for one hour. The temperature of the incubation was increased to 37°C, to optimize I-Sce I enzymatic activity, for an additional 2 hours. Linearization of the plasmid bacterial DNA circle by the restriction enzyme resulted in its rapid degradation by exonucleases in bacteria. The mini-circle expression cassette becomes the only episomal circular DNA and is easily isolated by an affinity purification Molecular Therapy Volume 9, Supplement 1, May 2004
Copyright The American Society of Gene Therapy