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DNA lipoplex
LIPID MEDIATED GENE TRANSFER (PEG) shielding has been proposed as a way to alleviate this effect, but was still found unsatisfactory in most instances for systemic administration. We demonstrate here that the insertion of anionic functions between the lipid part and the PEG, at a correct distance to favor electrostatic interactions with the outer cationic layer of the lipoplexes, provides not only a decrease in the mean peripheral charge of the lipoplex ( z potential), but also a greater colloidal stability of the particles in the presence of serum. Transfection in the lung is also decreased with negatively charged PEG shielding, although no significant changes are observed in the tumor. This encouraging new approach should consequently be combined with active extra-cellular receptor targeting to achieve the desired delivery of the therapeutic DNA to tumor tissues.
476. Human Mesenchymal Stem Cells Genetically Modified To Express High Levels of Erythropoietin through Transfer of an Artificial Chromosome Engineered Using the ACE System Sandra Vanderbyl,1 Neil MacDonald,1 Tom Stodola,1 Adele Telenius,1 Sandra Stewart,1 Harry C. Ledebur, Jr.,1 Carl F. Perez.1 1 Chromos Molecular Systems, Inc., Burnaby, BC, Canada. The ACE System is a versatile, reliable system for genetically modifying cell therapies, generating transgenic animals, and engineering mammalian cells for high expression of a recombinant protein. Key components of the ACE System include an artificial chromosome (Platform ACE) encoding multiple (> 50) DNA sitespecific integration sites (acceptor sites); a targeting vector (ACE Targeting Vector) encoding both a Platform ACE-specific DNA donor site and the therapeutic gene of interest; and a proprietary DNA recombinase (ACE Integrase) that catalyzes the site-specific recombination of ACE Targeting Vectors onto the Platform ACE. ACEs are a promising means of genetically modifying and engineering cells for cell therapy, as they are stably maintained, autonomous, non-integrating, easily purified by flow cytometry (de Jong et al, 1999) and readily transfected into a variety of cell types, including human adult stem cells (deJong et al, 2001; Vanderbyl et al, 2001; Vanderbyl et al, Stem Cells in Press). In this study, we engineered a Platform ACE to encode both the humanized renilla green fluorescent protein (hrGFP) and the human erythropoietin (epo) genes. A Platform ACE, carried in a CHO cell line, was first loaded with an ACE Targeting Vector encoding the hrGFP gene. Systematic analysis of drug resistant colonies by PCR, FISH and flow cytometry demonstrated that greater than 80% of the analyzed clones contained hrGFP-loaded ACEs and expressed GFP. Subsequent loading of the hrGFP-ACE with a second ACE Targeting Vector encoding the human epo gene yielded epo-loaded and expressing ACEs in approximately 83% of the GFP+ colonies. The resultant hrGFP-epo-ACEs were isolated and purified by flow cytometry and transfected into adult human mesenchymal stem cells (hMSCs) using cationic agents. The transfected hMSC population, which was enriched to 20% GFP+ cells by flow cytometry, secreted epo in the range of 50-100 IU/106cells/day. These levels of epo expression are comparable to those attained with multiple cycles of epo-retroviral transductions in MSCs (Bartholomew et al, 2001). Currently we are implanting epo-expressing hMSCs into NOD/ SCID mice and will monitor hematocrits over extended periods of time. We believe the above study will provide seminal data demonstrating that artificial chromosomes engineered using the ACE System will provide a safe and viable approach for ex vivo genemodified cell therapies.
Molecular Therapy Volume 9, Supplement 1, May 2004 Copyright © The American Society of Gene Therapy
477. New Pyridinium Cationic Lipids for Gene Delivery Synthesized Via Pyrylium Salts Marc A. Ilies,1 Betty H. Johnson,2 William A. Seitz,1 Edward L. Ezell,2 Aaron Miller,2 E. Brad Thompson,2 Alexandru T. Balaban.1 1 MARS, Texas A&M University at Galveston, Galveston, TX; 2 Department of Human Biological Chemistry and Genetics, University of Texas Medical Branch at Galveston, Galveston, TX. A new approach for generating cationic lipids for gene delivery bearing pyridinium polar heads was described recently [1]. It is based on the reaction of pyrylium salts with primary amines thus generating the polar head and the linker of the non-viral transfection vector in a single, high yield step. The versatility of the synthesis allows the choice of a large variety of aliphatic or aromatic linkers, yielding cationic lipids such as 1–4, some of which act better than commercial transfection systems. The method also allows the easy access to the class of gemini surfactants such as 5 and congeners, and the facile choice of the counterion of the final positively charged species. Additionally, the use of styryl pyrylium salts can yield fluorescent transfection systems and membrane markers such as 6. Detailed insights into their chemical synthesis and transfection properties on different tumor cell lines are presented, together with a complete structure - activity relationship study for each class of lipophilic pyridinium compounds. [1] M.A. Ilies, W.A. Seitz, M.T. Caproiu, M. Wentz, R.E. Garfield, A.T. Balaban, Eur. J. Org. Chem. (2003) 2645-2655.
478. Preinjection of Free DiC14-Amidine Liposome and Dexamethasone Synergistically Enhance the Intravenous Transfection Efficiency of Cationic Lipid/DNA Lipoplex Caroline Lonez, Jean-Marie Ruysschaert, Abdelatif Elouahabi. 1 Structure and Function of Biological Membranes, Free University of Brussels, Brussels, Belgium. Cationic lipids represent a safe alternative to viral vectors for gene therapy applications but their use is still limited by a low in vivo gene transfer efficiency. We report here that cationic lipidmediated intravenous transfection efficiency can be improved by a single preinjection of free diC14-amidine liposomes or of the steroidal anti-inflammatory drug dexamethasone (Dex). This improvement is dose dependent. Surprisingly, the transfection activity of the complex was enhanced by approximately 24 fold when both diC14-amidine free liposomes and Dex were preinjected, at a doses where individually they have no effect. Synergy between diC14-amidine free liposomes and Dex improved also the intravenous transfection efficiency of DOTAP/DNA lipoplex demonstrating that the method is applicable to other lipoplexes. These finding suggest preinjection of diC14amidine and dexamethasone as a way to improve the gene transfer efficiency and reduce the toxicity of liposomes.
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476. Human Mesenchymal Stem Cells Genetically Modified To Express High Levels of Erythropoietin through Transfer of an Artificial Chromosome Engineered Using the ACE System Sandra Vanderbyl,1 Neil MacDonald,1 Tom Stodola,1 Adele Telenius,1 Sandra Stewart,1 Harry C. Ledebur, Jr.,1 Carl F. Perez.1 1 Chromos Molecular Systems, Inc., Burnaby, BC, Canada. The ACE System is a versatile, reliable system for genetically modifying cell therapies, generating transgenic animals, and engineering mammalian cells for high expression of a recombinant protein. Key components of the ACE System include an artificial chromosome (Platform ACE) encoding multiple (> 50) DNA sitespecific integration sites (acceptor sites); a targeting vector (ACE Targeting Vector) encoding both a Platform ACE-specific DNA donor site and the therapeutic gene of interest; and a proprietary DNA recombinase (ACE Integrase) that catalyzes the site-specific recombination of ACE Targeting Vectors onto the Platform ACE. ACEs are a promising means of genetically modifying and engineering cells for cell therapy, as they are stably maintained, autonomous, non-integrating, easily purified by flow cytometry (de Jong et al, 1999) and readily transfected into a variety of cell types, including human adult stem cells (deJong et al, 2001; Vanderbyl et al, 2001; Vanderbyl et al, Stem Cells in Press). In this study, we engineered a Platform ACE to encode both the humanized renilla green fluorescent protein (hrGFP) and the human erythropoietin (epo) genes. A Platform ACE, carried in a CHO cell line, was first loaded with an ACE Targeting Vector encoding the hrGFP gene. Systematic analysis of drug resistant colonies by PCR, FISH and flow cytometry demonstrated that greater than 80% of the analyzed clones contained hrGFP-loaded ACEs and expressed GFP. Subsequent loading of the hrGFP-ACE with a second ACE Targeting Vector encoding the human epo gene yielded epo-loaded and expressing ACEs in approximately 83% of the GFP+ colonies. The resultant hrGFP-epo-ACEs were isolated and purified by flow cytometry and transfected into adult human mesenchymal stem cells (hMSCs) using cationic agents. The transfected hMSC population, which was enriched to 20% GFP+ cells by flow cytometry, secreted epo in the range of 50-100 IU/106cells/day. These levels of epo expression are comparable to those attained with multiple cycles of epo-retroviral transductions in MSCs (Bartholomew et al, 2001). Currently we are implanting epo-expressing hMSCs into NOD/ SCID mice and will monitor hematocrits over extended periods of time. We believe the above study will provide seminal data demonstrating that artificial chromosomes engineered using the ACE System will provide a safe and viable approach for ex vivo genemodified cell therapies.
Molecular Therapy Volume 9, Supplement 1, May 2004 Copyright © The American Society of Gene Therapy
477. New Pyridinium Cationic Lipids for Gene Delivery Synthesized Via Pyrylium Salts Marc A. Ilies,1 Betty H. Johnson,2 William A. Seitz,1 Edward L. Ezell,2 Aaron Miller,2 E. Brad Thompson,2 Alexandru T. Balaban.1 1 MARS, Texas A&M University at Galveston, Galveston, TX; 2 Department of Human Biological Chemistry and Genetics, University of Texas Medical Branch at Galveston, Galveston, TX. A new approach for generating cationic lipids for gene delivery bearing pyridinium polar heads was described recently [1]. It is based on the reaction of pyrylium salts with primary amines thus generating the polar head and the linker of the non-viral transfection vector in a single, high yield step. The versatility of the synthesis allows the choice of a large variety of aliphatic or aromatic linkers, yielding cationic lipids such as 1–4, some of which act better than commercial transfection systems. The method also allows the easy access to the class of gemini surfactants such as 5 and congeners, and the facile choice of the counterion of the final positively charged species. Additionally, the use of styryl pyrylium salts can yield fluorescent transfection systems and membrane markers such as 6. Detailed insights into their chemical synthesis and transfection properties on different tumor cell lines are presented, together with a complete structure - activity relationship study for each class of lipophilic pyridinium compounds. [1] M.A. Ilies, W.A. Seitz, M.T. Caproiu, M. Wentz, R.E. Garfield, A.T. Balaban, Eur. J. Org. Chem. (2003) 2645-2655.
478. Preinjection of Free DiC14-Amidine Liposome and Dexamethasone Synergistically Enhance the Intravenous Transfection Efficiency of Cationic Lipid/DNA Lipoplex Caroline Lonez, Jean-Marie Ruysschaert, Abdelatif Elouahabi. 1 Structure and Function of Biological Membranes, Free University of Brussels, Brussels, Belgium. Cationic lipids represent a safe alternative to viral vectors for gene therapy applications but their use is still limited by a low in vivo gene transfer efficiency. We report here that cationic lipidmediated intravenous transfection efficiency can be improved by a single preinjection of free diC14-amidine liposomes or of the steroidal anti-inflammatory drug dexamethasone (Dex). This improvement is dose dependent. Surprisingly, the transfection activity of the complex was enhanced by approximately 24 fold when both diC14-amidine free liposomes and Dex were preinjected, at a doses where individually they have no effect. Synergy between diC14-amidine free liposomes and Dex improved also the intravenous transfection efficiency of DOTAP/DNA lipoplex demonstrating that the method is applicable to other lipoplexes. These finding suggest preinjection of diC14amidine and dexamethasone as a way to improve the gene transfer efficiency and reduce the toxicity of liposomes.
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