Key issues in non-viral gene delivery

Pouton, Colin W.; Seymour, Len

doi:10.1016/s0169-409x(98)00048-9

Cited by 288 publications

(221 citation statements)

References 95 publications

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“…The penetration of DNA to the nucleus is considered to be one of the major obstacles for nonviral gene delivery. 29 However, in the case of ODS-based polyplexes, the majority of the labeled plasmids are observed inside the cell nucleus at these time points.…”

Section: Intracellular Traffickingmentioning

confidence: 92%

Novel dextran–spermine conjugates as transfecting agents: comparing water-soluble and micellar polymers

et al. 2004

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Recently, a novel cationic polymer, dextran-spermine (D-SPM) was developed for gene delivery. An efficient transfection was obtained using this polycation for a variety of genes and cell lines in serum-free or serum-poor medium. However, transfection using the water-soluble D-SPMbased polyplexes decreased with increasing serum concentration in cell culture in a concentration-dependent manner, reaching 95% inhibition at 50% serum in the cell growth medium. In order to overcome this obstacle, oleyl derivatives of D-SPM (which form micelles in aqueous phase) were synthesized at 1, 10, and 20 mol% of oleyl moiety to polymer e-NH 2 to form N-oleyl-D-SPM (ODS). Polyplexes based on ODS transfected well in medium containing 50% serum. Comparison with polyplexes based on well-established polymers (branched and linear polyethyleneimine) and with DOTAP/Cholesterol lipoplexes showed that regarding b-galactosidase transgene expression level and cytotoxicity in tissue culture, the D-SPM and ODS compare well with the above polyplexes and lipoplexes. Intracellular trafficking using FITC-labeled ODS and Rhodamine-labeled pGeneGrip plasmid cloned with hBMP2 monitored by confocal microscopy revealed that during the transfection process the fluorescent-labeled polymer concentrates in the Golgi apparatus and around the nucleus, while the cell cytoplasm was free of fluorescent particles, suggesting that the polyplexes move in the cell toward the nucleus by vesicular transport through the cytoplasm and not by a random diffusion. We found that the plasmids penetrate the cell nucleus without the polymer. Preliminary results in zebra fish and mice demonstrate the potential of ODS to serve as an efficient nonviral vector for in vivo transfection. Gene Therapy (2005) 12, 494-503.

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Section: Intracellular Traffickingmentioning

confidence: 92%

Novel dextran–spermine conjugates as transfecting agents: comparing water-soluble and micellar polymers

et al. 2004

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“…Active transport and passive diffusion are both potential contributors to the transport of fluorescently labeled, plasmid DNA nucleotides across the cellular barriers and are characterized by the rate constants in Table 4. The conversion of rate constants to reflect active and passive transport was performed using Equations (11) and (14). The active transport rate constant across the medium/cytoplasm (mc) barrier was positive, implying net import of plasmid DNA nucleotides into the cell cytoplasm (k mc act ¼ 0:63 h À1 ).…”

Section: Hela Cellsmentioning

confidence: 99%

“…Additional composite rate constants, K, defined in Table 1, were also calculated from the initially obtained optimized rates. The diffusive and active rate constants are then found from the previous work by using Equations (11) and (14). These constants are presented in terms of volumes to facilitate comparison among experiments.…”

Section: Computational Compartmental Modelmentioning

confidence: 99%

“…Physical methods include ballistics (the 'gene gun'), 9 electroporation, 10 ultrasound, 11 hydrodynamic pressure changes, 12 and jet injection. 13 Biochemical delivery methods such as lipid formulations, cationic lipid chemistry, 14,15 DNA condensation, addition of glycosylated moieties, 16 and use of synthetic peptides to act as NLSs have also been studied. The cellular mechanisms of these delivery methods are, generally, not well understood, and the fate of the plasmids has also not been comprehensively quantified or evaluated.…”

Section: Introductionmentioning

confidence: 99%

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A model for the analysis of nonviral gene therapy

et al. 2003

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Further understanding of the mechanisms involved in cellular and intracellular delivery of transgene is needed to produce clinical applications of gene therapy. The compartmental and computational model designed in this work is integrated with data from previous experiments to quantitatively estimate rate constants of plasmid translocation across cellular barriers in transgene delivery in vitro. The experimental conditions between two cellular studies were held constant, varying only the cell type, to investigate how the rates differed between cell lines. Two rate constants were estimated per barrier for active transport and passive diffusion. Translocation rates of intact plasmid across the cytoplasmic and nuclear barriers varied between cell lines. CV1 cells were defined by slower rates (0.23 h À1 cytoplasmic, 0.08 h -1 nuclear) than those of the HeLa cells (1.87 h À1 cytoplasmic, 0.45 h À1 nuclear). The nuclear envelope was identified as a rate-limiting barrier by comparing the rate of intact plasmid translocation at each barrier. Slower intact plasmid translocation in CV1 cells was correlated with a reduced absolute capacity for transgene efficiency in comparison with HeLa cells. HeLa cells were three times more efficient than CV1 cells at producing green fluorescent protein per intact plasmid delivered to the nucleus. Mathematical modeling coordinated with experimental studies can provide detailed, quantitative understanding of nonviral gene therapy.

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“…18,19 Success of nonviral gene delivery depends on the type of carrier materials to bind plasmid DNA and facilitate the cell-specific uptake of carrier-DNA complex. [20][21][22] In addition, the in vivo gene expression is greatly influenced by the body distribution of the carrier-DNA complex intravenously injected. The gene transfection with naked plasmid DNA always shows low efficiency in vivo although it is simple and safe.…”

Section: Introductionmentioning

confidence: 99%

Dextran–spermine polycation: an efficient nonviral vector for in vitro and in vivo gene transfection

et al. 2004

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Dextran-spermine cationic polysaccharide was prepared by means of reductive amination between oxidized dextran and the natural oligoamine spermine. The formed Schiff-base imine-based conjugate was reduced with borohydride to obtain the stable amine-based conjugate. The transfection efficiency of the synthetic dextran-spermine was assessed in vitro on HEK293 and NIH3T3 cell lines and found to be as high as the DOTAP/Chol 1/1 lipid-based transfection reagent. Modification of the dextran-spermine polycation with polyethylene glycol resulted in high transfection yield in serum-rich medium. Intramuscular injection in mice of dextran-spermine-pSV-LacZ complex induced high local gene expression compared to low expression of the naked DNA. Intravenous injection of a dispersion of the dextranspermine-pSV-LacZ complex resulted with no expression in all examined organs. When the partially PEGylated dextranspermine-pSV-LacZ complex was intravenously applied, a high gene expression was detected mainly in the liver. Preliminary targeting studies indicated that the PEGylated dextran-spermine-pSV-LacZ complex bound to galactose receptor of liver parenchymal cells rather than the mannose receptor of liver nonparenchymal cells. This work offers a new biodegradable polycation based on natural components, which is capable of transfecting cells and tissues in vitro and in vivo.

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Key issues in non-viral gene delivery

Cited by 288 publications

References 95 publications

Novel dextran–spermine conjugates as transfecting agents: comparing water-soluble and micellar polymers

Novel dextran–spermine conjugates as transfecting agents: comparing water-soluble and micellar polymers

A model for the analysis of nonviral gene therapy

Dextran–spermine polycation: an efficient nonviral vector for in vitro and in vivo gene transfection

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