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486. Structure-Activity Relationship of a New Class of Polyamines
CHEMICAL AND MOLECULAR CONJUGATES II 486. Structure-Activity Relationship of a New Class of Polyamines
Xiang Gao,1 Ramalinga Kuruba,1 Damodaran K. Achary,2 Billy W. Day,1 Dexi Liu,1 Song Li.1 1 Center for Pharmacogenetics, Department of Pharmaceutical Sciences, University of Pittsburgh School of Pharmacy, Pittsburgh, PA; 2Department of Chemistry, University of Pittsburgh School of Arts and Sciences, Pittsburgh, PA. Cationic polymers constitute an important class of gene transfer agent. However, a detailed structure-activity relationship for these agents is still murky. We have synthesized a panel of structurally related amine-containing polycations whose overall backbone structures resemble polyethylenimines. These are co-polymers synthesized from a di -or oligoamine and a dichlorine cross-linker by alkylation. The choice of amines with different numbers of amine groups, the length between these amine groups and different substitutions, and the chain length and terminal group of the side chain in the cross-linkers provide great diversity to the resulting polycations. The reaction conditions determine the MWs of the final products. These polycations exhibited great variations in their transfection activity and cytotoxicity levels. Polymers with moderate MW gave the best overall performance both in transfection efficiency and cytotoxicity, while polymers with high MWs had high toxicity and limited useful dosages for transfection. Polymers with increased linear charge density from oligoamines did not increase the overall transfection efficiency vs. a polymer of lesser density from a simple diamine, but required lower polymer to DNA ratios for peak level of transfection. The cross-linkers with different side chain lengths drastically affected the transfection and toxicity of the polymers, with medium length being the optimal structure. Finally, inclusion of reducible disulfide bonds to the polymer backbone increased transfection activity and reduced cytotoxicity. Overall, our studies reveal some structure-activity relationships of these new polycations in gene delivery.
487. Microarray Analysis of Intracellular Signaling Pathways in Nonviral Gene Transfer
Gina Boanca,1 Angela K. Pannier.1 1 Biological Systems Engineering, University of Nebraska-Lincoln, Lincoln, NE. The use of gene delivery in therapeutic applications, including gene therapy to treat genetic deficiencies or tissue engineering matrices for the treatment of organ loss and failure, has been limited due to challenges with current delivery systems. Nonviral vectors, which typically involve electrostatic complexation of cationic polymers or lipids with DNA, are significantly less efficient than viral vectors, but offer advantages of low toxicity and immunogenicity, lack pathogenicity, and are easy to produce with greater control and flexibility, making these vectors attractive alternatives to viruses. To date, most efforts to understand and improve the efficiency of nonviral delivery have focused on altering the physicochemical properties of delivery systems and developing new delivery strategies. However, the exact mechanisms involved in gene delivery are poorly defined, including the intracellular signaling pathways activated in response to gene transfer, which govern trafficking of the DNA. The importance of cell signaling in achieving successful nonviral gene transfer has not been thoroughly examined, though it clearly plays a role in regulation of cellular responses and may affect the ability of cells to become transfected. We have used microarray analysis to identify signaling pathways that are modulated during nonviral gene transfer. Nonviral DNA complexes (composed of cationic lipids or polymers complexed with plasmid DNA encoding for GFP) were delivered to HEK293T human embryonic kidney epithelial cells, a widely used cell line in transfection experiments. Flow cytometry was then used to sort GFP-positive cells, allowing isolation of a S188
population of transfected cells. These cells were then lysed and their mRNA collected and purified using standard techniques. The RNA samples were then hybridized to Affymetrix GeneChip expression arrays. Control and experimental samples were hybridized to separate chips, in triplicate. Expression patterns were compared between transfected and nontransfected samples, which revealed several key differential gene expression profiles regulated by nonviral gene transfer. With a greater understanding of key signaling pathways involved in gene delivery, we hope to understand the mechanisms that render cells responsive to DNA transfer to develop more efficient nonviral delivery schemes.
488. Cellular Delivery of Plasmid DNA and siRNA to Adherent and Suspension Cancer Cells by Sendai Virosomes
Jom Ee Baek,1 Jung Seok Kim,1 Yeon Kyung Lee,1 Sang Il Park,1 Hwa Yon Jung,1 Keun Sik Kim,2 Yong Serk Park.1 1 Biomedical Laboratory Science, Yonsei University, Wonju, Gangwon, Korea, Republic of; 2Biomedical Sciences, Youngdong University, Woungdong, Chungbuk, Korea, Republic of. Previously, we have shown that the Sendai F/HN virosomes are an effective gene delivery system for in vitro and in vivo transgene expression. The Sendai virosomes consist of two different glycoproteins, hemagglutininneuraminidase (HN) and fusion protein (F) which are required for binding to cell surface and cell fusion, respectively. In this study, we constructed two different types of virosomes, the so-called cationic Sendai F/HN virosomes (CSVs) and protamine sulfate-condensed cationic Sendai F/HN virosomes (PCSVs). The plasmid DNA or siRNA was complexed with the CSVs or PCSVs, and then transferred to adherent cancer cells (293 and HeLa) and suspension cancer cells (Jurkat). Generally, the PCSVs were more effective in delivery of pDNA to the cultured cancer cells than the CSVs and conventional cationic lipoplexes. Meanwhile, according to the FACS analysis the CSVs exhibited more effective delivery of siRNA to Jurkat cells, one of the toughest cells for transfection, than the PCSVs. The effective intracellular uptake and endosomal escape of pDNA (or siRNA) transferred with the PCSVs and CSVs were confirmed by confocal-microscopic analysis. From these experimental results, it can be concluded that the PCSVs and CSVs would be widely utilized as an alternative gene (or siRNA) delivery system for various types of cells including suspension cancer cells.
489. A Novel Mixing Device for the Reproducible Manufacture of Non-Viral Gene Therapy Formulations
Lee A. Davies,1,4 Graciela A. Nunez-Alonso,1,4 Henry L. Hebel,2 Ron K. Scheule,3 Seng H. Cheng,3 Deborah R. Gill,1,4 Stephen C. Hyde.1,4 1 Gene Medicine Group, NDCLS, University of Oxford, Oxford, United Kingdom; 2VGXI Inc., The Woodlands, TX; 3Genzyme Corporation, Framingham, MA; 4UK Cystic Fibrosis Gene Therapy Consortium, Edinburgh/London/Oxford, United Kingdom.
The generation of non-viral gene therapy formulations requires the complexation of negatively charged plasmid DNA (pDNA) with cationic gene transfer agents (GTAs) such as lipids, polymers and peptides. Within the laboratory, small volumes of reagent are often prepared by stepwise addition of one reagent to the other. However, this technique is inappropriate for the production of larger amounts of material required for clinical applications because incomplete or variable mixing associated with larger volumes can significantly affect both the physical characteristics and the in vivo performance of the complexes. We have developed a pneumatic mixing device that allows the reliable and reproducible mixing of large volumes Molecular Therapy Volume 17, Supplement 1, May 2009 Copyright © The American Society of Gene Therapy
Xiang Gao,1 Ramalinga Kuruba,1 Damodaran K. Achary,2 Billy W. Day,1 Dexi Liu,1 Song Li.1 1 Center for Pharmacogenetics, Department of Pharmaceutical Sciences, University of Pittsburgh School of Pharmacy, Pittsburgh, PA; 2Department of Chemistry, University of Pittsburgh School of Arts and Sciences, Pittsburgh, PA. Cationic polymers constitute an important class of gene transfer agent. However, a detailed structure-activity relationship for these agents is still murky. We have synthesized a panel of structurally related amine-containing polycations whose overall backbone structures resemble polyethylenimines. These are co-polymers synthesized from a di -or oligoamine and a dichlorine cross-linker by alkylation. The choice of amines with different numbers of amine groups, the length between these amine groups and different substitutions, and the chain length and terminal group of the side chain in the cross-linkers provide great diversity to the resulting polycations. The reaction conditions determine the MWs of the final products. These polycations exhibited great variations in their transfection activity and cytotoxicity levels. Polymers with moderate MW gave the best overall performance both in transfection efficiency and cytotoxicity, while polymers with high MWs had high toxicity and limited useful dosages for transfection. Polymers with increased linear charge density from oligoamines did not increase the overall transfection efficiency vs. a polymer of lesser density from a simple diamine, but required lower polymer to DNA ratios for peak level of transfection. The cross-linkers with different side chain lengths drastically affected the transfection and toxicity of the polymers, with medium length being the optimal structure. Finally, inclusion of reducible disulfide bonds to the polymer backbone increased transfection activity and reduced cytotoxicity. Overall, our studies reveal some structure-activity relationships of these new polycations in gene delivery.
487. Microarray Analysis of Intracellular Signaling Pathways in Nonviral Gene Transfer
Gina Boanca,1 Angela K. Pannier.1 1 Biological Systems Engineering, University of Nebraska-Lincoln, Lincoln, NE. The use of gene delivery in therapeutic applications, including gene therapy to treat genetic deficiencies or tissue engineering matrices for the treatment of organ loss and failure, has been limited due to challenges with current delivery systems. Nonviral vectors, which typically involve electrostatic complexation of cationic polymers or lipids with DNA, are significantly less efficient than viral vectors, but offer advantages of low toxicity and immunogenicity, lack pathogenicity, and are easy to produce with greater control and flexibility, making these vectors attractive alternatives to viruses. To date, most efforts to understand and improve the efficiency of nonviral delivery have focused on altering the physicochemical properties of delivery systems and developing new delivery strategies. However, the exact mechanisms involved in gene delivery are poorly defined, including the intracellular signaling pathways activated in response to gene transfer, which govern trafficking of the DNA. The importance of cell signaling in achieving successful nonviral gene transfer has not been thoroughly examined, though it clearly plays a role in regulation of cellular responses and may affect the ability of cells to become transfected. We have used microarray analysis to identify signaling pathways that are modulated during nonviral gene transfer. Nonviral DNA complexes (composed of cationic lipids or polymers complexed with plasmid DNA encoding for GFP) were delivered to HEK293T human embryonic kidney epithelial cells, a widely used cell line in transfection experiments. Flow cytometry was then used to sort GFP-positive cells, allowing isolation of a S188
population of transfected cells. These cells were then lysed and their mRNA collected and purified using standard techniques. The RNA samples were then hybridized to Affymetrix GeneChip expression arrays. Control and experimental samples were hybridized to separate chips, in triplicate. Expression patterns were compared between transfected and nontransfected samples, which revealed several key differential gene expression profiles regulated by nonviral gene transfer. With a greater understanding of key signaling pathways involved in gene delivery, we hope to understand the mechanisms that render cells responsive to DNA transfer to develop more efficient nonviral delivery schemes.
488. Cellular Delivery of Plasmid DNA and siRNA to Adherent and Suspension Cancer Cells by Sendai Virosomes
Jom Ee Baek,1 Jung Seok Kim,1 Yeon Kyung Lee,1 Sang Il Park,1 Hwa Yon Jung,1 Keun Sik Kim,2 Yong Serk Park.1 1 Biomedical Laboratory Science, Yonsei University, Wonju, Gangwon, Korea, Republic of; 2Biomedical Sciences, Youngdong University, Woungdong, Chungbuk, Korea, Republic of. Previously, we have shown that the Sendai F/HN virosomes are an effective gene delivery system for in vitro and in vivo transgene expression. The Sendai virosomes consist of two different glycoproteins, hemagglutininneuraminidase (HN) and fusion protein (F) which are required for binding to cell surface and cell fusion, respectively. In this study, we constructed two different types of virosomes, the so-called cationic Sendai F/HN virosomes (CSVs) and protamine sulfate-condensed cationic Sendai F/HN virosomes (PCSVs). The plasmid DNA or siRNA was complexed with the CSVs or PCSVs, and then transferred to adherent cancer cells (293 and HeLa) and suspension cancer cells (Jurkat). Generally, the PCSVs were more effective in delivery of pDNA to the cultured cancer cells than the CSVs and conventional cationic lipoplexes. Meanwhile, according to the FACS analysis the CSVs exhibited more effective delivery of siRNA to Jurkat cells, one of the toughest cells for transfection, than the PCSVs. The effective intracellular uptake and endosomal escape of pDNA (or siRNA) transferred with the PCSVs and CSVs were confirmed by confocal-microscopic analysis. From these experimental results, it can be concluded that the PCSVs and CSVs would be widely utilized as an alternative gene (or siRNA) delivery system for various types of cells including suspension cancer cells.
489. A Novel Mixing Device for the Reproducible Manufacture of Non-Viral Gene Therapy Formulations
Lee A. Davies,1,4 Graciela A. Nunez-Alonso,1,4 Henry L. Hebel,2 Ron K. Scheule,3 Seng H. Cheng,3 Deborah R. Gill,1,4 Stephen C. Hyde.1,4 1 Gene Medicine Group, NDCLS, University of Oxford, Oxford, United Kingdom; 2VGXI Inc., The Woodlands, TX; 3Genzyme Corporation, Framingham, MA; 4UK Cystic Fibrosis Gene Therapy Consortium, Edinburgh/London/Oxford, United Kingdom.
The generation of non-viral gene therapy formulations requires the complexation of negatively charged plasmid DNA (pDNA) with cationic gene transfer agents (GTAs) such as lipids, polymers and peptides. Within the laboratory, small volumes of reagent are often prepared by stepwise addition of one reagent to the other. However, this technique is inappropriate for the production of larger amounts of material required for clinical applications because incomplete or variable mixing associated with larger volumes can significantly affect both the physical characteristics and the in vivo performance of the complexes. We have developed a pneumatic mixing device that allows the reliable and reproducible mixing of large volumes Molecular Therapy Volume 17, Supplement 1, May 2009 Copyright © The American Society of Gene Therapy