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484. Safety of ΦC31 Integrase Expression in Mouse Liver Following Hydrodynamic Injection
484. Safety of q>C31 Integrase Expression in Mouse Liver Following Hydrodynamic Injection
Christopher L. Chavez, Lauren E. Woodard , Annahita Keravala, Vanessa Gabrovsky. 'Genetics, Stanford. Stanford. CA.
tpC3l intcgrasc, a site-specific integrase from the bacteriophage tpC31, catalyzes chromosomal integration of plasmid DNA at ' pseudo' attP sites in mammalian genomes. tpC3l integrase has been used to achieve integration and long-term, robust expression of therapeutic genes in many tissues, including mouse liver. No immune reaction or tumors have been observed in these studies. We are undertaking additional studies to examine more thoroughly the safety of using tpC31 integrasc in gene therapy of the liver. The predicted short duration of integrase expression would be a safety feature by limiting exposure of cells to the reeombinase. In order to test this hypothesis, we are determining the longevity ofintegrase protein afler hydrodynamic injection of an integrase expression vector in the mouse liver. The longevity of integrase expression will be determined by removing the liver at various time points, isolating total protein for Western blotting from half, and sectioning the remaining half for immunohistochemistry with an antibody detecting
485. Analysis of the Transfection Behaviour of Supercoiled Plasmid DNA Concatemers In Vitro Christof'Maucksch .P Florian Hoffmann,' Martin Schleef,' Manish K. Aneja, ' Markus Elflnger;'? Dominic Hartl,' Carsten Rudolph.l-' 'Department ofPediatrics, Ludwig-Ma;rimilians-University. Munich, Germany; lDepartment ofPharmacy, Free University ofBerlin, Berlin . Germany; "Plasmidliactory GmbH & Co KG. Plasmidliactory GmbH & Co KG. Bielefeld. Germany.
Naked supercoiled plasmid DNA is widely used in nonviral gene delivery in vitro and in vivo. However, it has not yet been systematically examined, ifthe arrangement ofconcatemers may be beneficial for gene expression ofnaked supereoiled plasmid DNA. In this study we compared a supercoiled 4.7 kb pEGFP monomer and its 9.4 kb dimerie coneatemer, which carries two repeats of identical pEGFP monomer molecules linked to each other head-to-tail. Naked pDNA was transfected into Jurkat T cells by electroporation. The number and mean fluorescent intensity of EGFP expressing cells was analyzed by flow cytometry (FACS) 24 hrs after electroporation. In addition, we determined the relative amounts ofpDNA delivered to the nucleus by using fluorescently TOTO-I labeled pDNA to establish a correlation between intranuclear pDNA and gene expression. The mean average diameter of both plasm ids was measured by dynamic light scattering. Transfections were performed using either equal Molecular Therapy Volume 15.Supplement I. ~br Copyright © The American Soc iety of Gene Therapy
2007
numbers of EGFP gene copies or plasmid molecules. The nuclei of these cells were isolated and the relative intranuclear amounts ofpDNA expressed as the TOTO-I MFI were measured by FACS. Transfection ofthe 80 nm pEGFP-monomer resulted in significantly 1.54-fold higher number of EGFP expressing cells compared to the 150 nm dimeric pEGFP, when equimolar numbers of EGFP gene copies were transfcctcd, Although the EGFP-MFI was not different, relatively less EGFP gene copies entered the nucleus when EGFPmonomer compared with dimer was transfected as indicated by the 1'01'0-1 MFI ofnuclei. The relative gene copy expression efficiency indicated by the ratio of the EGFP MFI of the transfected cells to 1'01'0-1 MFI per nucleus was 1.5-fold greater for pEGFP-dimer than for pEGFP-monomer. Analogous observations were made when equal numbers of plasmid molecule were transfcctcd, These observations suggest that the eoncatemer arrangement increases relative gene expression efficiency, whereas plasmid size is important for cell and nucleus entry afler e1ectroporation. We suggest using preferably small supercoiled plasmid concatemers as the ideal plasmid vectors for non viral gene therapy.
486. Rapid Deployment Plasmid Production Process, Combining Inducible High Yield FedBatch Fermentation Process with Novel Autolytic Plasmid DNA Purification Aaron Carnes, 1 James Williams,' Jeremy Luke; Sarah Langtry,' Clague Hodgson.' [Research and Development. Nature Technology Corporation. Lincoln. NE. DNA vaccines and gene medicines, derived from bacterial plasmids, are emerging as an important new elass of pharmaceuticals. They may allow the manufacturer to bypass years of development for the production of efficacious vaccines, and literally create new vaccine entities in days and mass prod uce vaccines in 2-3 weeks for rapid deployment against new biological agents. However, the challenges of producing plasmid DNA at an industrial scale are well known: low bioreactor yields, scaling up alkaline lysis, clearance of host RNA and chromosomal DNA, and avoiding the use of animal sourced products. This limits their utility to meet cost and capacity needs for existing plasmid applications, or to rapidly produce kilograms of plasmid DNA for pandemic vaccination. The development of an inducible fed-batch fermentation process that dramatically increases volumetric yield and specific plasmid yield, while maintaining or enhancing plasmid integrity has been the first achievement toward these goals. This inducible process utilizes commercially available media that we designed specifically for plasmid production. The process consists ofan initial biomass accumulation phase , followed by a plasmid accumulation phase. The plasmid is stably maintained at low levels during a period ofnutrient restricted growth and reduced temperature (30°C), and then the temperature is increased (37-42°C) to induce plasmid amplification. Typically, the specific plasmid yield increases over a period of up to 15 hours following temperature up-shill. Volumetric yields exceeding 2. I g plasmid DNA/L with specific yields of 43 mg/gm dry cell weight have been achieved with this process, and this process has been successfully scaled up to the 300 L scale for GMP production. To address downstream purification challenges, we have successfully demonstrated feasibility of a cost effective, streamlined, and simple purification process that eliminates costly alkaline or heat lysis steps and the associated toxic waste streams. We developed autolytic E. coli host strains with integrated chimeric nucleases. In this purification process, autolysis is performed at moderate temperatures, without alkaline lysis or the addition of lysozyme. The endogenously produced chimeric nuclease then selectively degrades host nucleic acids. Purification of the plasmid is completed in a chromatography-free process, with high final product recoveries. Sl87
Christopher L. Chavez, Lauren E. Woodard , Annahita Keravala, Vanessa Gabrovsky. 'Genetics, Stanford. Stanford. CA.
tpC3l intcgrasc, a site-specific integrase from the bacteriophage tpC31, catalyzes chromosomal integration of plasmid DNA at ' pseudo' attP sites in mammalian genomes. tpC3l integrase has been used to achieve integration and long-term, robust expression of therapeutic genes in many tissues, including mouse liver. No immune reaction or tumors have been observed in these studies. We are undertaking additional studies to examine more thoroughly the safety of using tpC31 integrasc in gene therapy of the liver. The predicted short duration of integrase expression would be a safety feature by limiting exposure of cells to the reeombinase. In order to test this hypothesis, we are determining the longevity ofintegrase protein afler hydrodynamic injection of an integrase expression vector in the mouse liver. The longevity of integrase expression will be determined by removing the liver at various time points, isolating total protein for Western blotting from half, and sectioning the remaining half for immunohistochemistry with an antibody detecting
485. Analysis of the Transfection Behaviour of Supercoiled Plasmid DNA Concatemers In Vitro Christof'Maucksch .P Florian Hoffmann,' Martin Schleef,' Manish K. Aneja, ' Markus Elflnger;'? Dominic Hartl,' Carsten Rudolph.l-' 'Department ofPediatrics, Ludwig-Ma;rimilians-University. Munich, Germany; lDepartment ofPharmacy, Free University ofBerlin, Berlin . Germany; "Plasmidliactory GmbH & Co KG. Plasmidliactory GmbH & Co KG. Bielefeld. Germany.
Naked supercoiled plasmid DNA is widely used in nonviral gene delivery in vitro and in vivo. However, it has not yet been systematically examined, ifthe arrangement ofconcatemers may be beneficial for gene expression ofnaked supereoiled plasmid DNA. In this study we compared a supercoiled 4.7 kb pEGFP monomer and its 9.4 kb dimerie coneatemer, which carries two repeats of identical pEGFP monomer molecules linked to each other head-to-tail. Naked pDNA was transfected into Jurkat T cells by electroporation. The number and mean fluorescent intensity of EGFP expressing cells was analyzed by flow cytometry (FACS) 24 hrs after electroporation. In addition, we determined the relative amounts ofpDNA delivered to the nucleus by using fluorescently TOTO-I labeled pDNA to establish a correlation between intranuclear pDNA and gene expression. The mean average diameter of both plasm ids was measured by dynamic light scattering. Transfections were performed using either equal Molecular Therapy Volume 15.Supplement I. ~br Copyright © The American Soc iety of Gene Therapy
2007
numbers of EGFP gene copies or plasmid molecules. The nuclei of these cells were isolated and the relative intranuclear amounts ofpDNA expressed as the TOTO-I MFI were measured by FACS. Transfection ofthe 80 nm pEGFP-monomer resulted in significantly 1.54-fold higher number of EGFP expressing cells compared to the 150 nm dimeric pEGFP, when equimolar numbers of EGFP gene copies were transfcctcd, Although the EGFP-MFI was not different, relatively less EGFP gene copies entered the nucleus when EGFPmonomer compared with dimer was transfected as indicated by the 1'01'0-1 MFI ofnuclei. The relative gene copy expression efficiency indicated by the ratio of the EGFP MFI of the transfected cells to 1'01'0-1 MFI per nucleus was 1.5-fold greater for pEGFP-dimer than for pEGFP-monomer. Analogous observations were made when equal numbers of plasmid molecule were transfcctcd, These observations suggest that the eoncatemer arrangement increases relative gene expression efficiency, whereas plasmid size is important for cell and nucleus entry afler e1ectroporation. We suggest using preferably small supercoiled plasmid concatemers as the ideal plasmid vectors for non viral gene therapy.
486. Rapid Deployment Plasmid Production Process, Combining Inducible High Yield FedBatch Fermentation Process with Novel Autolytic Plasmid DNA Purification Aaron Carnes, 1 James Williams,' Jeremy Luke; Sarah Langtry,' Clague Hodgson.' [Research and Development. Nature Technology Corporation. Lincoln. NE. DNA vaccines and gene medicines, derived from bacterial plasmids, are emerging as an important new elass of pharmaceuticals. They may allow the manufacturer to bypass years of development for the production of efficacious vaccines, and literally create new vaccine entities in days and mass prod uce vaccines in 2-3 weeks for rapid deployment against new biological agents. However, the challenges of producing plasmid DNA at an industrial scale are well known: low bioreactor yields, scaling up alkaline lysis, clearance of host RNA and chromosomal DNA, and avoiding the use of animal sourced products. This limits their utility to meet cost and capacity needs for existing plasmid applications, or to rapidly produce kilograms of plasmid DNA for pandemic vaccination. The development of an inducible fed-batch fermentation process that dramatically increases volumetric yield and specific plasmid yield, while maintaining or enhancing plasmid integrity has been the first achievement toward these goals. This inducible process utilizes commercially available media that we designed specifically for plasmid production. The process consists ofan initial biomass accumulation phase , followed by a plasmid accumulation phase. The plasmid is stably maintained at low levels during a period ofnutrient restricted growth and reduced temperature (30°C), and then the temperature is increased (37-42°C) to induce plasmid amplification. Typically, the specific plasmid yield increases over a period of up to 15 hours following temperature up-shill. Volumetric yields exceeding 2. I g plasmid DNA/L with specific yields of 43 mg/gm dry cell weight have been achieved with this process, and this process has been successfully scaled up to the 300 L scale for GMP production. To address downstream purification challenges, we have successfully demonstrated feasibility of a cost effective, streamlined, and simple purification process that eliminates costly alkaline or heat lysis steps and the associated toxic waste streams. We developed autolytic E. coli host strains with integrated chimeric nucleases. In this purification process, autolysis is performed at moderate temperatures, without alkaline lysis or the addition of lysozyme. The endogenously produced chimeric nuclease then selectively degrades host nucleic acids. Purification of the plasmid is completed in a chromatography-free process, with high final product recoveries. Sl87