- Email: [email protected]
526. Induction of Apoptosis in Melanoma Cell Lines by the Noxa Gene
DNA VECTOROLOGY sites. For the purpose of gene therapy, it appears desirable to obtain integration at only one or a small set of safe, well-expressed locations in the genome. In a previous study we demonstrated that by using a genetic screening assay in E. coli, we could create mutant forms of phiC31 integrase with enhanced preference for a pre-existing favored integration site in the human genome. However, this enzyme displayed a low overall integration efficiency. To create phiC31 integrase mutants with enhanced functionality, we devised a screen in mammalian cells to identify mutants with increased recombination efficiency at the attP site. We isolated a collection of such mutants, and combination of several of them led to the identification of an enzyme that mediated the majority of integration events at a single location, at a pre-integrated attP site. This enzyme may be useful for the rapid creation of cell lines with integration at a fixed location. The study also provided proof-of-principle for creation of integrases highly specific for endogenous sequences, a project is underway in the lab. Other mutants from the screen showed an elevated integration frequency at endogenous pseudo attP sites and are being tested in animals as improved gene therapy tools. We have also undertaken the structural characterization of phiC31 integrase. The location of the DNA-binding domain is being investigated by creating deletion mutants and examining them for att site binding with gel mobility shift assays. The increased efficiency and specificity attainable via mutagenesis and protein design will further enhance the utility of phiC31 integrase as a valuable tool in gene therapy. Michele P Calos is a co-founder and consultant for Poetic Genetics, LLC, which has licensed the phiC31 integrase technology.
526. Induction of Apoptosis in Melanoma Cell Lines by the Noxa Gene Mohamed Hassan,1 Amine Alaoui,1 Alireza Mirmohammadsadegh,1 Alessandra Marini,1 Sandeep Nambiar,1 Ulrich R. Hengge.1 1 Dept. of Dermatology, Heinrich-Heine-Universit, Duesseldorf, Germany. Background: Melanoma is the most aggressive form of skin cancers and has shown a dramatic increase in incidence over the past decades. Although new therapies have become available, unfortunately advanced tumors are invariably resistant to conventional treatment due to a dysregulation of apoptosis. In the present study, we demonstrate the feasibility of electroporation of the BH3-only gene Noxa to induce apoptosis in melanoma cell lines. Material and Methods: Flow cytometric analysis, direct immunofluorescence (DIF), Western Blot. Results: The direct transfer of Noxa gene into the melanoma cell lines BLM and A375 promoted cell death in up to 95% as evidenced by annexin-V and JC-1 flow cytometry and immunofluorescence. The percentage of the resulting cell death correlated with the transfection efficiency. To investigate whether Noxa-induced cell death was mediated through an apoptotic mechanism, we examined PARP cleavage in transfected melanoma cell lines. Using immunoblotting, we could detect the Noxa protein (6 kDa) as well as the 85 kDa cleavage product of PARP 72h post transfection. Conclusion: Our data demonstrate the potential of the Noxa gene to overcome the resistance and to promote cell death in melanoma cell lines. Therefore, the targeted delivery of the pro-apoptotic Noxa gene using an adenoviral vector may become an attractive strategy for melanoma therapy.
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527. Pharmaceutical Plasmid Manufacturing: Development of Economical and Innovative Process Solutions Joachim Schorr,1 Markus Mueller,1 Juergen Hubbuch,2 Andreas Frerix.2 1 Research & Development, QIAGEN GmbH, Hilden, Germany; 2 BioSeparations Group, Research Center Juelich, Juelich, Germany. The commercialisation of plasmid DNA for gene therapy and DNA vaccination applications will be dependent on the availability of large scale manufacturing technology that allows reasonable Costof-Goods in a manufacturing scenario. Here, we present the outcome of process development studies based on the objective to design a price efficient process without compromising on product quality or compliance. Those studies lead to an economical and robust vacuum-based pre-clarification technique, able to remove the shear sensitive KDS precipitate from crude bacterial lysate before loading onto the QIAGEN TM anion exchange resin. A follow on one step chromatography run results in pharmaceutical grade DNA. We also have developed an Aqueous-Two-Phase-Separation (ATPS) step. Using ATPS can further refine the plasmid intermediate to even higher purity specifications for e.g. high-dosage plasmid applications. ATPS has economical benefits in the field of largescale plasmid manufacturing due to the unique selectivity achieved, combined with low cost of goods. The new ATPS-System is able to deplete an excess of open circular (oc) plasmid topoisomers and genomic host DNA from the plasmid preparation. The combination of ATPS and the QIAGEN one step anion exchange chromatography process enables pharmaceutical grade plasmid production at reduced time-in-the-facility, capital investment and product loss without any compromise on target specifications.
528. Significant Silencing Effect of a Multifunctional Envelope-Type Nano Device Encapsulating Antisense Oligodeoxynucleotide and Double-Stranded siRNA Kentaro Kogure,1,2 Yoshio Nakamura,1,2 Ikramy A. Khalil,1,2 Shiroh Futaki,3,4 Hideyoshi Harashima.1,2 1 Graduate School of Pharmaceutical Sciences, Hokkaido University, Sapporo, Hokkaido, Japan; 2CREST, Japan Science and Technology Agency (JST), Japan; 3Institute for Chemical Research, Kyoto University, Uji, Kyoto, Japan; 4PRESTO, Japan Science and Technology Agency (JST), Japan. For efficient gene delivery to the nucleus, non-viral vectors need to overcome several barriers such as the plasma membrane, the endosomal membrane and the nuclear membrane. To overcome these obstacles, it is necessary to equip the delivery system with various functional devices [Kamiya H., et al., Drug Discov. Today 21 (2003) 990]. However, it is difficult to package all these functional devices into a single system to exert each of their functions at the appropriate time and at the correct location. Thus, we proposed a new packaging concept, “Programmed Packaging”. This concept consists of three components: (1) A program to overcome all barriers. (2) Development of functional devices and their three dimensional assignment. (3) Nano-technology for assembling all devices into a nano-size structure. A multifunctional envelope-type nano device (MEND) was developed for use as an efficient non-viral system for the delivery of plasmid DNA (pDNA) using octaarginine (R8) as an internalizing ligand based on the Programmed Packaging [Kogure et al, J. Control.
Molecular Therapy Volume 13, Supplement 1, May 2006 Copyright The American Society of Gene Therapy
526. Induction of Apoptosis in Melanoma Cell Lines by the Noxa Gene Mohamed Hassan,1 Amine Alaoui,1 Alireza Mirmohammadsadegh,1 Alessandra Marini,1 Sandeep Nambiar,1 Ulrich R. Hengge.1 1 Dept. of Dermatology, Heinrich-Heine-Universit, Duesseldorf, Germany. Background: Melanoma is the most aggressive form of skin cancers and has shown a dramatic increase in incidence over the past decades. Although new therapies have become available, unfortunately advanced tumors are invariably resistant to conventional treatment due to a dysregulation of apoptosis. In the present study, we demonstrate the feasibility of electroporation of the BH3-only gene Noxa to induce apoptosis in melanoma cell lines. Material and Methods: Flow cytometric analysis, direct immunofluorescence (DIF), Western Blot. Results: The direct transfer of Noxa gene into the melanoma cell lines BLM and A375 promoted cell death in up to 95% as evidenced by annexin-V and JC-1 flow cytometry and immunofluorescence. The percentage of the resulting cell death correlated with the transfection efficiency. To investigate whether Noxa-induced cell death was mediated through an apoptotic mechanism, we examined PARP cleavage in transfected melanoma cell lines. Using immunoblotting, we could detect the Noxa protein (6 kDa) as well as the 85 kDa cleavage product of PARP 72h post transfection. Conclusion: Our data demonstrate the potential of the Noxa gene to overcome the resistance and to promote cell death in melanoma cell lines. Therefore, the targeted delivery of the pro-apoptotic Noxa gene using an adenoviral vector may become an attractive strategy for melanoma therapy.
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527. Pharmaceutical Plasmid Manufacturing: Development of Economical and Innovative Process Solutions Joachim Schorr,1 Markus Mueller,1 Juergen Hubbuch,2 Andreas Frerix.2 1 Research & Development, QIAGEN GmbH, Hilden, Germany; 2 BioSeparations Group, Research Center Juelich, Juelich, Germany. The commercialisation of plasmid DNA for gene therapy and DNA vaccination applications will be dependent on the availability of large scale manufacturing technology that allows reasonable Costof-Goods in a manufacturing scenario. Here, we present the outcome of process development studies based on the objective to design a price efficient process without compromising on product quality or compliance. Those studies lead to an economical and robust vacuum-based pre-clarification technique, able to remove the shear sensitive KDS precipitate from crude bacterial lysate before loading onto the QIAGEN TM anion exchange resin. A follow on one step chromatography run results in pharmaceutical grade DNA. We also have developed an Aqueous-Two-Phase-Separation (ATPS) step. Using ATPS can further refine the plasmid intermediate to even higher purity specifications for e.g. high-dosage plasmid applications. ATPS has economical benefits in the field of largescale plasmid manufacturing due to the unique selectivity achieved, combined with low cost of goods. The new ATPS-System is able to deplete an excess of open circular (oc) plasmid topoisomers and genomic host DNA from the plasmid preparation. The combination of ATPS and the QIAGEN one step anion exchange chromatography process enables pharmaceutical grade plasmid production at reduced time-in-the-facility, capital investment and product loss without any compromise on target specifications.
528. Significant Silencing Effect of a Multifunctional Envelope-Type Nano Device Encapsulating Antisense Oligodeoxynucleotide and Double-Stranded siRNA Kentaro Kogure,1,2 Yoshio Nakamura,1,2 Ikramy A. Khalil,1,2 Shiroh Futaki,3,4 Hideyoshi Harashima.1,2 1 Graduate School of Pharmaceutical Sciences, Hokkaido University, Sapporo, Hokkaido, Japan; 2CREST, Japan Science and Technology Agency (JST), Japan; 3Institute for Chemical Research, Kyoto University, Uji, Kyoto, Japan; 4PRESTO, Japan Science and Technology Agency (JST), Japan. For efficient gene delivery to the nucleus, non-viral vectors need to overcome several barriers such as the plasma membrane, the endosomal membrane and the nuclear membrane. To overcome these obstacles, it is necessary to equip the delivery system with various functional devices [Kamiya H., et al., Drug Discov. Today 21 (2003) 990]. However, it is difficult to package all these functional devices into a single system to exert each of their functions at the appropriate time and at the correct location. Thus, we proposed a new packaging concept, “Programmed Packaging”. This concept consists of three components: (1) A program to overcome all barriers. (2) Development of functional devices and their three dimensional assignment. (3) Nano-technology for assembling all devices into a nano-size structure. A multifunctional envelope-type nano device (MEND) was developed for use as an efficient non-viral system for the delivery of plasmid DNA (pDNA) using octaarginine (R8) as an internalizing ligand based on the Programmed Packaging [Kogure et al, J. Control.
Molecular Therapy Volume 13, Supplement 1, May 2006 Copyright The American Society of Gene Therapy