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559. Progress towards Clinically Viable Next Generation Liposome:Mu:DNA (LMD) Systems
LIPID MEDIATED GENE TRANSFER ² P. Erbacher, J-S Remy, J-P Behr. Gene Ther. 1996, 6, 138-145. ³S.L. Hart, C.V. Arancibia-Carcamo, M.A. Wolfert, C. Mailhos, N.J. O’Reily, R.R. Ali, C. Coutelle, A.J.T. George, R.P. Harbottle, A.M. Knight, D.F.P. Larkin, R.J. Levinsky, L.W. Seymour, A.J. Thrasher, C. Kinnon. Hum. Gene Ther. 1998, 9, 575-585. 4 R.P. Harbottle, R.G. Cooper, S.L. Hart, A. Ladhoff, T. McKay, A.M. Knight, E. Wagner, A.D. Miller, C. Coutelle. Hum. Gene Ther. 1998, 9, 1037-1047.
559. Progress towards Clinically Viable Next Generation Liposome:Mu:DNA (LMD) Systems Kostas Kostarelos,1 Andrew D. Miller.1 Imperial College Genetic Therapies Centre, Imperial College London, London, United Kingdom.
transfection efficiency. We further investigated effect of wortmannin, an inhibitor of phosphatidylinositol 3-kinase (PI 3-kinase), an enzyme involved in endocytotic process, on transfection efficiency in the above cell-types. Observed cell-specific inhibitions indicate a possible role of PI 3-kinase sensitive and insensitive endocytotic routes for the entry of lipid/DNA complexes in different cell lines. Quantitation of cell associated DNA, as measuremed by radioactive DNA, does not show correlation with the transfection efficiency. This lack of correlation was found, on investigation, to be due to cell-specific variations in non-specific adsorption of DNA. Using confocal microscopy, we have quantitated the internalized FITC labeled DNA.
1
The development of clinically viable, synthetic non-viral vector systems depends largely on the rational design of self-assembly complexes involving cationic lipids (cytofectins) and nucleic acids such as plasmid DNA. However, in spite of major problems emerging through the use of viral vector systems in clinical trials, synthetic non-viral vector systems have as yet been unable to capture sufficient clinical interest primarily due to a number of significant problems including poor mediation of gene expression in vivo and irreproducible biological profiles. We have been developing a bottom-up, logical chemical biology approach to these and other problems of synthetic non-viral vector systems based around the creation of a non-viral vector platform system known as liposome:mu:DNA (LMD) that can now be upgraded by a series of modular adaptations leading towards the ultimate goal of clinically viable, synthetic non-viral vector systems. First generation LMD is prepared from DC-Chol/DOPE cationic liposomes, mu peptide of the adenovirus core, and plasmid DNA. This has proven to be a robust platform able to give reproducible transfection results in mouse lung in vivo at a level equivalent to “gold standard” GL-67 based non-viral vector systems. Here we will present a number of recent modular adaptations to first generation LMD in persuit of improved second-generation LMD systems. These modular adaptations include (a) the inclusion of neoglycolipids into the LMD outer coat as a means to provide some measure of biological stability whilst retaining transfection efficacy; (b) the use of integrin-targeting lipopeptides (tenascin peptides) to initiate targeted-LMD transfections in vitro; (c) the introduction of novel DOPE analogues to engineer the outer coat of the LMD vector system to create LMD systems with comparable transfection efficacies in vitro but with substantially reduced positive charge. Some or all of these adaptations should help to enhance the efficacy of LMD system transfections of mouse lung in vivo.
560. Validating the Role of Endocytosis in Lipofection T. K. Prasad,1 N. M. Rao.1 1 Centre for Cellular and Molecular Biology, Hyderabad, Andhra Pradesh, India. Transfectability of a variety of cell lines to a given transfection protocol is extremely variable. The cell biological basis for this variability between cell lines is not clearly understood. The predominant pathway for entry of lipid:DNA complexes into the cells is considered to be the endocytotic pathway. We investigated dependence of transfection efficiency on endocytotic rate in eleven different cell lines. Selected eleven cell lines differ more than 50 fold to a single transfection protocol. Endocytotic rates of 11 cell lines were determined using horseradish peroxidase as a marker. We observed a significant positive correlation (P< 0.001) between the transfection efficiency and the endocytotic rates, which suggests endocytotic entry of lipoplexes into the cells has strong bearing on S218
561. Synthesis and Biological Evaluation of Alpha 9 Beta 1 Integrin Targeted Non-Viral Gene Delivery System Jodie E. Waterhouse,1 Michael R. Jorgensen,1 Richard P. Harbottle,2 Kostas Kostarelos,1 Andrew D. Miller.1 1 Genetic Therapies Centre, Chemistry, Imperial College London, London, United Kingdom; 2Gene Therapy Group, Cell and Molecular Biology, Imperial College London, London, United Kingdom. One of the main problems facing gene therapy is the ability to target specific cells. Our research is a synthetic based approach to this problem. LMD (liposome:mu:DNA) is our current non-viral vector platform technology that is an efficient non-viral gene delivery system. LMD is the end result of the formulation of two lipids, CDAN or DC-Chol combined with DOPE to generate liposomes, which are then complexed with plasmid DNA pre-condensed by mu peptide. Currently, this technology has no targeting ability; therefore to initiate this idea, novel molecules, (lipopeptides) have been synthesised covalently linking cholesterol to an integrin targeting peptide, PLAEIDGIELA. This peptide sequence is taken from the protein tenascin and is a recognised targeting ligand known to bind to cell integrins, alpha 9 beta 1. These receptors are commonly found in lung epithelial cells, so a possible application of this work would be for the treatment of cystic fibrosis. Lipopeptides are incorporated into LMD and LD systems (up to 5 mol %) to form the basis of targeted non-viral vector systems. Initial results show enhancement of transfection but with inconsistent targeting effects. Evaluation of the peptide and the lipopeptides alone demonstrates their abilities to target cells selectively through competitive cell binding assays. These lipopeptides are found to be slightly toxic, but once incorporated into a liposome, this toxicity diminishes. We are currently exploring different liposome formulations to reduce the non-specific effects that appear to be the basis of the inconsistent targeting.
562. Antisense Phosphothioate Oligonucleotides Access Via RNA Matrix to mRNA Target. Downregulation of 5HT1a Gene Fuxin Shi,1 Willy H. Visser,1 Eric Ronken,2 Natasja M. J. D. Jong,2 Dick Hoekstra.1 1 Membrane Cell Biology, University of Groningen, Groningen, Groningen, Netherlands; 2Research Laboratories, Solvay Pharmaceuticals, Weesp, Amesterdam, Netherlands. Successful application of antisense oligonucleotides (ODNs) in cell biology and therapy will depend on ease of design and exquisite target specificity. Part of this goal includes a need to carefully development a means for efficient (intra)cellular delivery in conjunction with a detailed understanding of the mechanism of antisense action. The ability of an antisense sequence to modulate protein expression can be affected by the nature of the target cell, efficiency of intracellular delivery, and ODN stability. Here, we Molecular Therapy Vol. 7, No. 5, May 2003, Part 2 of 2 Parts
Copyright © The American Society of Gene Therapy
559. Progress towards Clinically Viable Next Generation Liposome:Mu:DNA (LMD) Systems Kostas Kostarelos,1 Andrew D. Miller.1 Imperial College Genetic Therapies Centre, Imperial College London, London, United Kingdom.
transfection efficiency. We further investigated effect of wortmannin, an inhibitor of phosphatidylinositol 3-kinase (PI 3-kinase), an enzyme involved in endocytotic process, on transfection efficiency in the above cell-types. Observed cell-specific inhibitions indicate a possible role of PI 3-kinase sensitive and insensitive endocytotic routes for the entry of lipid/DNA complexes in different cell lines. Quantitation of cell associated DNA, as measuremed by radioactive DNA, does not show correlation with the transfection efficiency. This lack of correlation was found, on investigation, to be due to cell-specific variations in non-specific adsorption of DNA. Using confocal microscopy, we have quantitated the internalized FITC labeled DNA.
1
The development of clinically viable, synthetic non-viral vector systems depends largely on the rational design of self-assembly complexes involving cationic lipids (cytofectins) and nucleic acids such as plasmid DNA. However, in spite of major problems emerging through the use of viral vector systems in clinical trials, synthetic non-viral vector systems have as yet been unable to capture sufficient clinical interest primarily due to a number of significant problems including poor mediation of gene expression in vivo and irreproducible biological profiles. We have been developing a bottom-up, logical chemical biology approach to these and other problems of synthetic non-viral vector systems based around the creation of a non-viral vector platform system known as liposome:mu:DNA (LMD) that can now be upgraded by a series of modular adaptations leading towards the ultimate goal of clinically viable, synthetic non-viral vector systems. First generation LMD is prepared from DC-Chol/DOPE cationic liposomes, mu peptide of the adenovirus core, and plasmid DNA. This has proven to be a robust platform able to give reproducible transfection results in mouse lung in vivo at a level equivalent to “gold standard” GL-67 based non-viral vector systems. Here we will present a number of recent modular adaptations to first generation LMD in persuit of improved second-generation LMD systems. These modular adaptations include (a) the inclusion of neoglycolipids into the LMD outer coat as a means to provide some measure of biological stability whilst retaining transfection efficacy; (b) the use of integrin-targeting lipopeptides (tenascin peptides) to initiate targeted-LMD transfections in vitro; (c) the introduction of novel DOPE analogues to engineer the outer coat of the LMD vector system to create LMD systems with comparable transfection efficacies in vitro but with substantially reduced positive charge. Some or all of these adaptations should help to enhance the efficacy of LMD system transfections of mouse lung in vivo.
560. Validating the Role of Endocytosis in Lipofection T. K. Prasad,1 N. M. Rao.1 1 Centre for Cellular and Molecular Biology, Hyderabad, Andhra Pradesh, India. Transfectability of a variety of cell lines to a given transfection protocol is extremely variable. The cell biological basis for this variability between cell lines is not clearly understood. The predominant pathway for entry of lipid:DNA complexes into the cells is considered to be the endocytotic pathway. We investigated dependence of transfection efficiency on endocytotic rate in eleven different cell lines. Selected eleven cell lines differ more than 50 fold to a single transfection protocol. Endocytotic rates of 11 cell lines were determined using horseradish peroxidase as a marker. We observed a significant positive correlation (P< 0.001) between the transfection efficiency and the endocytotic rates, which suggests endocytotic entry of lipoplexes into the cells has strong bearing on S218
561. Synthesis and Biological Evaluation of Alpha 9 Beta 1 Integrin Targeted Non-Viral Gene Delivery System Jodie E. Waterhouse,1 Michael R. Jorgensen,1 Richard P. Harbottle,2 Kostas Kostarelos,1 Andrew D. Miller.1 1 Genetic Therapies Centre, Chemistry, Imperial College London, London, United Kingdom; 2Gene Therapy Group, Cell and Molecular Biology, Imperial College London, London, United Kingdom. One of the main problems facing gene therapy is the ability to target specific cells. Our research is a synthetic based approach to this problem. LMD (liposome:mu:DNA) is our current non-viral vector platform technology that is an efficient non-viral gene delivery system. LMD is the end result of the formulation of two lipids, CDAN or DC-Chol combined with DOPE to generate liposomes, which are then complexed with plasmid DNA pre-condensed by mu peptide. Currently, this technology has no targeting ability; therefore to initiate this idea, novel molecules, (lipopeptides) have been synthesised covalently linking cholesterol to an integrin targeting peptide, PLAEIDGIELA. This peptide sequence is taken from the protein tenascin and is a recognised targeting ligand known to bind to cell integrins, alpha 9 beta 1. These receptors are commonly found in lung epithelial cells, so a possible application of this work would be for the treatment of cystic fibrosis. Lipopeptides are incorporated into LMD and LD systems (up to 5 mol %) to form the basis of targeted non-viral vector systems. Initial results show enhancement of transfection but with inconsistent targeting effects. Evaluation of the peptide and the lipopeptides alone demonstrates their abilities to target cells selectively through competitive cell binding assays. These lipopeptides are found to be slightly toxic, but once incorporated into a liposome, this toxicity diminishes. We are currently exploring different liposome formulations to reduce the non-specific effects that appear to be the basis of the inconsistent targeting.
562. Antisense Phosphothioate Oligonucleotides Access Via RNA Matrix to mRNA Target. Downregulation of 5HT1a Gene Fuxin Shi,1 Willy H. Visser,1 Eric Ronken,2 Natasja M. J. D. Jong,2 Dick Hoekstra.1 1 Membrane Cell Biology, University of Groningen, Groningen, Groningen, Netherlands; 2Research Laboratories, Solvay Pharmaceuticals, Weesp, Amesterdam, Netherlands. Successful application of antisense oligonucleotides (ODNs) in cell biology and therapy will depend on ease of design and exquisite target specificity. Part of this goal includes a need to carefully development a means for efficient (intra)cellular delivery in conjunction with a detailed understanding of the mechanism of antisense action. The ability of an antisense sequence to modulate protein expression can be affected by the nature of the target cell, efficiency of intracellular delivery, and ODN stability. Here, we Molecular Therapy Vol. 7, No. 5, May 2003, Part 2 of 2 Parts
Copyright © The American Society of Gene Therapy