The Experts below are selected from a list of 255 Experts worldwide ranked by ideXlab platform
Patrick Midoux - One of the best experts on this subject based on the ideXlab platform.
-
Intracellular processing and stability of DNA complexed with histidylated Polylysine conjugates.
Journal of Gene Medicine, 2002Co-Authors: Christine Gonçalves, Chantal Pichon, Brigitte Guérin, Patrick MidouxAbstract:Background Glycosylated Polylysines and histidylated Polylysines complexed with plasmid DNA (pDNA) were proposed to develop polymer-based gene delivery systems. The present work has been undertaken in two steps to study the uptake and the intracellular processing of pDNA, which are still poorly understood in the polyfection pathway. Methods and results The kinetics of the uptake and the intracellular processing of pDNA complexed with lactosylated Polylysine, histidylated Polylysine or histidylated Polylysine bearing lactosyl residues (polyplexes) into a CF human airway epithelial cell line were assessed by flow cytometry and confocal microscopy. Complexes formed from histidylated Polylysine, even though they were less taken up by cells, show better transfection efficiency with compared with lactosylated complexes. Lactosylated polymers segregated more rapidly when compared with non-lactosylated polymers into compartments different from those containing pDNA on internalization. Intracellular location and pH measurements indicated that polymers ended up in compartments of pH ∼6.2 while pDNA reached less acidic compartments of pH ∼6.6. These compartments did not contain the LAMP-1 lysosomal marker. Conclusions The present study exhibits that, upon internalization, pDNA and Polylysine conjugates underwent segregation with a rate depending on the Polylysine substitution and polymer degradation. The better transfection efficiency of polyplexes with histidylated Polylysine can be ascribed to their prolonged stability inside the endocytic vesicles that likely favored the pDNA escape in the cytosol. Copyright © 2002 John Wiley & Sons, Ltd.
-
Histidylated Polylysine as a synthetic vector for gene transfer into immortalized cystic fibrosis airway surface and airway gland serous cells
The Journal of Gene Medicine, 2000Co-Authors: Isabelle Fajac, Michel Monsigny, Chantal Pichon, Pascale Briand, J.c Allo, Marc Merten, Evelyne Souil, Catherine Figarella, Patrick MidouxAbstract:We recently designed a cationic polymer called histidylated Polylysine made of Polylysine partially substituted with histidyl residues which become protonated at slightly acidic pH. This polymer is thought to induce the leakage of acidic vesicles containing plasmid/histidylated Polylysine complexes.
-
Efficient Gene Transfer into Human Normal and Cystic Fibrosis Tracheal Gland Serous Cells with Synthetic Vectors
American Journal of Respiratory Cell and Molecular Biology, 2000Co-Authors: J.c Allo, Patrick Midoux, Michel Monsigny, Pascale Briand, Marc Merten, Evelyne Souil, Joanna Lipecka, Catherine Figarella, Isabelle FajacAbstract:Submucosal gland serous cells are believed to play a major role in the physiopathology of cystic fibrosis (CF) and may represent an important target for CF gene therapy. We have studied the efficiency of reporter gene transfer into immortalized normal (MM-39) and CF (CF-KM4) human airway epithelial gland serous cells using various synthetic vectors: glycosylated Polylysines (glycofectins), polyethylenimine (PEI) (25 and 800 kD), lipofectin, and lipofectAMINE. In both cell lines, a high luciferase activity was achieved with various glycofectins, with PEI 25 kD, and with lipofectAMINE. After three transfections applied daily using alpha-glycosylated Polylysine, 20% of the cells were transfected. At 24 h after CF transmembrane conductance regulator (CFTR) gene transfer into CF-KM4 cells using alpha-glycosylated Polylysine, the immunolocalization of CFTR was analyzed by laser scanning confocal microscopy and the transgenic CFTR was detected by an intense labeling of the plasma membrane. The presence of membrane lectins, i. e., cell surface receptors binding oligosaccharides, was also examined on MM-39 and CF-KM4 cells by assessing the binding and uptake of fluorescein-labeled neoglycoproteins and fluorescein-labeled glycoplexes (glycofectins complexed to plasmid DNA). Among all the neoglycoproteins and glycoplexes tested, those bearing alpha-mannosylated derivatives were most efficiently taken up by both normal and CF gland serous cells. However, alpha-mannosylated Polylysine was quite inefficient for gene transfer, indicating that the efficiency of gene transfer is determined both by the uptake of the complexes and also by their intracellular trafficking. Moreover, our results show that an efficient in vitro gene transfer was achieved in human airway gland serous cells with the same synthetic vectors described to efficiently transfect human airway surface epithelial cells.
-
Sugar-Mediated Uptake of Glycosylated Polylysines and Gene Transfer into Normal and Cystic Fibrosis Airway Epithelial Cells
Human Gene Therapy, 1999Co-Authors: Isabelle Fajac, Michel Monsigny, Pascale Briand, Patrick MidouxAbstract:We have examined the membrane lectin expressed by immortalized normal and cystic fibrosis (CF) airway epithelial cells, using fluorescein-labeled neoglycoproteins; the uptake of plasmid DNA using fluoresceinylated glycoplexes (plasmid/glycosylated Polylysine complexes); and the efficiency of gene transfer when glycosylated Polylysines and glycosylated, partially gluconoylated Polylysines were used as vectors. The most efficient uptake of neoglycoproteins by normal and CF cells was obtained with mannosylated BSA (bovine serum albumin). Similarly, the most efficient uptake of plasmid DNA was obtained with glycoplexes bearing alpha-D-Man residues. Surprisingly, glycoplexes bearing alpha-D-Man residues were poorly efficient for gene transfer into normal and CF cells. The highest luciferase activity was achieved with lactosylated Polylysine- and beta-D-GlcNAc-substituted gluconoylated Polylysine as vectors. Gene transfer efficiency obtained with gluconoylated Polylysine bearing beta-D-GlcNAc residues was similar to that observed with polyethylenimine (PEI; 25 and 800 kDa) and 10-fold higher than that observed with lipofectin and LipofectAMINE. These results suggest that the transfection efficiency with glycoplexes is not determined only by the specificity of the lectin expressed at the cell surface membrane but also by intracellular trafficking of the glycoplexes, which could be mediated by lectins present inside the cells.
-
Specific Gene Transfer Based on Biotinylated and Gluconoylated Polylysine/Plasmid Complexes
Drug Delivery, 1997Co-Authors: Patrick Erbacher, Annie Claude Roche, Michel Monsigny, Patrick MidouxAbstract:AbstractComplexes formed between plasmids and Polylysine derivatives bearing recognition signals are the basis of nonviral vehicles suitable for gene delivery into eukaryotic cells by a receptor-mediated endocytosis process. We used an alternative procedure with the aim of achieving noncovalent attachment of recognition signals to plasmids. Biotinylated Polylysine/DNA complexes were made between a plasmid and biotinylated Polylysine by electrostatic interactions, and then the DNA complex was lactosylated via streptavidin bridges in order to target the galactose-specific membrane lectin of human hepatoma (HepG2) cells. HepG2 cells were efficiently transfected in a sugar-dependent manner with a polymer/DNA complex lactosylated with either biotinylated and lactosylated bovine serum albumin or lactosylated streptavidin. In addition, reduction of the number of remaining cationic charges, by partial gluconoylation of biotinylated Polylysine, increased the transfection efficiency of HepG2 cells compared with tha...
Michel Monsigny - One of the best experts on this subject based on the ideXlab platform.
-
Histidylated Polylysine as a synthetic vector for gene transfer into immortalized cystic fibrosis airway surface and airway gland serous cells
The Journal of Gene Medicine, 2000Co-Authors: Isabelle Fajac, Michel Monsigny, Chantal Pichon, Pascale Briand, J.c Allo, Marc Merten, Evelyne Souil, Catherine Figarella, Patrick MidouxAbstract:We recently designed a cationic polymer called histidylated Polylysine made of Polylysine partially substituted with histidyl residues which become protonated at slightly acidic pH. This polymer is thought to induce the leakage of acidic vesicles containing plasmid/histidylated Polylysine complexes.
-
Efficient Gene Transfer into Human Normal and Cystic Fibrosis Tracheal Gland Serous Cells with Synthetic Vectors
American Journal of Respiratory Cell and Molecular Biology, 2000Co-Authors: J.c Allo, Patrick Midoux, Michel Monsigny, Pascale Briand, Marc Merten, Evelyne Souil, Joanna Lipecka, Catherine Figarella, Isabelle FajacAbstract:Submucosal gland serous cells are believed to play a major role in the physiopathology of cystic fibrosis (CF) and may represent an important target for CF gene therapy. We have studied the efficiency of reporter gene transfer into immortalized normal (MM-39) and CF (CF-KM4) human airway epithelial gland serous cells using various synthetic vectors: glycosylated Polylysines (glycofectins), polyethylenimine (PEI) (25 and 800 kD), lipofectin, and lipofectAMINE. In both cell lines, a high luciferase activity was achieved with various glycofectins, with PEI 25 kD, and with lipofectAMINE. After three transfections applied daily using alpha-glycosylated Polylysine, 20% of the cells were transfected. At 24 h after CF transmembrane conductance regulator (CFTR) gene transfer into CF-KM4 cells using alpha-glycosylated Polylysine, the immunolocalization of CFTR was analyzed by laser scanning confocal microscopy and the transgenic CFTR was detected by an intense labeling of the plasma membrane. The presence of membrane lectins, i. e., cell surface receptors binding oligosaccharides, was also examined on MM-39 and CF-KM4 cells by assessing the binding and uptake of fluorescein-labeled neoglycoproteins and fluorescein-labeled glycoplexes (glycofectins complexed to plasmid DNA). Among all the neoglycoproteins and glycoplexes tested, those bearing alpha-mannosylated derivatives were most efficiently taken up by both normal and CF gland serous cells. However, alpha-mannosylated Polylysine was quite inefficient for gene transfer, indicating that the efficiency of gene transfer is determined both by the uptake of the complexes and also by their intracellular trafficking. Moreover, our results show that an efficient in vitro gene transfer was achieved in human airway gland serous cells with the same synthetic vectors described to efficiently transfect human airway surface epithelial cells.
-
Sugar-Mediated Uptake of Glycosylated Polylysines and Gene Transfer into Normal and Cystic Fibrosis Airway Epithelial Cells
Human Gene Therapy, 1999Co-Authors: Isabelle Fajac, Michel Monsigny, Pascale Briand, Patrick MidouxAbstract:We have examined the membrane lectin expressed by immortalized normal and cystic fibrosis (CF) airway epithelial cells, using fluorescein-labeled neoglycoproteins; the uptake of plasmid DNA using fluoresceinylated glycoplexes (plasmid/glycosylated Polylysine complexes); and the efficiency of gene transfer when glycosylated Polylysines and glycosylated, partially gluconoylated Polylysines were used as vectors. The most efficient uptake of neoglycoproteins by normal and CF cells was obtained with mannosylated BSA (bovine serum albumin). Similarly, the most efficient uptake of plasmid DNA was obtained with glycoplexes bearing alpha-D-Man residues. Surprisingly, glycoplexes bearing alpha-D-Man residues were poorly efficient for gene transfer into normal and CF cells. The highest luciferase activity was achieved with lactosylated Polylysine- and beta-D-GlcNAc-substituted gluconoylated Polylysine as vectors. Gene transfer efficiency obtained with gluconoylated Polylysine bearing beta-D-GlcNAc residues was similar to that observed with polyethylenimine (PEI; 25 and 800 kDa) and 10-fold higher than that observed with lipofectin and LipofectAMINE. These results suggest that the transfection efficiency with glycoplexes is not determined only by the specificity of the lectin expressed at the cell surface membrane but also by intracellular trafficking of the glycoplexes, which could be mediated by lectins present inside the cells.
-
Specific Gene Transfer Based on Biotinylated and Gluconoylated Polylysine/Plasmid Complexes
Drug Delivery, 1997Co-Authors: Patrick Erbacher, Annie Claude Roche, Michel Monsigny, Patrick MidouxAbstract:AbstractComplexes formed between plasmids and Polylysine derivatives bearing recognition signals are the basis of nonviral vehicles suitable for gene delivery into eukaryotic cells by a receptor-mediated endocytosis process. We used an alternative procedure with the aim of achieving noncovalent attachment of recognition signals to plasmids. Biotinylated Polylysine/DNA complexes were made between a plasmid and biotinylated Polylysine by electrostatic interactions, and then the DNA complex was lactosylated via streptavidin bridges in order to target the galactose-specific membrane lectin of human hepatoma (HepG2) cells. HepG2 cells were efficiently transfected in a sugar-dependent manner with a polymer/DNA complex lactosylated with either biotinylated and lactosylated bovine serum albumin or lactosylated streptavidin. In addition, reduction of the number of remaining cationic charges, by partial gluconoylation of biotinylated Polylysine, increased the transfection efficiency of HepG2 cells compared with tha...
-
The reduction of the positive charges of Polylysine by partial gluconoylation increases the transfection efficiency of Polylysine/DNA complexes
Biochimica et Biophysica Acta, 1997Co-Authors: Patrick Erbacher, Annie Claude Roche, Michel Monsigny, Patrick MidouxAbstract:A Polylysine partially substituted with polyhydroxyalkanoyl residues and specially with gluconoyl residues was developed in order to increase the transfection efficiency by decreasing the strength of the electrostatic interactions between the DNA and the cationic polymer. Partially gluconoylated Polylysine/DNA complexes were more easily dissociated in solution and their transfection efficiency in the presence of chloroquine, evaluated with HepG2 cells, a human hepatocarcinoma line, was higher when 43 +/- 4% of the epsilon-amino groups of Polylysine were blocked with gluconoyl residues. Partially gluconoylated Polylysine/plasmid complexes were efficient in transfecting different adherent as well as non-adherent cell lines. Partially gluconoylated Polylysine formed highly soluble (above 100 micrograms/ml in DNA) complexes with DNA plasmids. In addition, partially gluconoylated Polylysine bearing few lactosyl residues increased the transfection efficiency of HepG2 cells which express a galactose-specific membrane lectin.
Isabelle Fajac - One of the best experts on this subject based on the ideXlab platform.
-
Intracellular rate-limiting steps of gene transfer using glycosylated Polylysines in cystic fibrosis airway epithelial cells
Gene Therapy, 2002Co-Authors: S Grosse, Pascale Briand, A Tremeau-bravard, Y Aron, Isabelle FajacAbstract:To identify the intracellular barriers to efficient gene transfer, we studied the intracellular trafficking of biotinylated plasmid DNA complexed with either fluorescein-conjugated lactosylated or mannosylated Polylysine by confocal microscopy. Both are known to be taken up by cystic fibrosis airway epithelial cells (ΣCFTE29o− cells), but their gene transfer efficiencies differ markedly: lactosylated Polylysine is the most efficient glycosylated Polylysine while mannosylated Polylysine is quite inefficient for gene transfer. Mannosylated complexes appeared to stay longer in endosomes labeled by anti-transferrin receptor antibody than lactosylated complexes (from 30 min to 3 h and from 10 min to 30 min, respectively). At 24 h, higher percentages of mannosylated than lactosylated complexes were localized inside lysosomes labeled by anti-LAMP-1 antibody (41.8 ± 6.6% versus 19.8 ± 5.2%, respectively, P < 0.05). Between 30 min and 8 h, complexes reached the nuclei labeled by anti-lamin A/C antibody and no difference was observed between the nuclear amounts of mannosylated and lactosylated complexes. However, as analyzed by a nuclease S1 transcription assay, initiation of transcription was prevented when plasmid DNA was complexed to mannosylated Polylysine. Our results indicate that the major limiting steps for mannosylated versus lactosylated Polylysine transfer of plasmid DNA are delayed exit from endosomes, high accumulation in lysosomes and limited transcription of the complexed plasmid DNA.
-
Histidylated Polylysine as a synthetic vector for gene transfer into immortalized cystic fibrosis airway surface and airway gland serous cells
The Journal of Gene Medicine, 2000Co-Authors: Isabelle Fajac, Michel Monsigny, Chantal Pichon, Pascale Briand, J.c Allo, Marc Merten, Evelyne Souil, Catherine Figarella, Patrick MidouxAbstract:We recently designed a cationic polymer called histidylated Polylysine made of Polylysine partially substituted with histidyl residues which become protonated at slightly acidic pH. This polymer is thought to induce the leakage of acidic vesicles containing plasmid/histidylated Polylysine complexes.
-
Efficient Gene Transfer into Human Normal and Cystic Fibrosis Tracheal Gland Serous Cells with Synthetic Vectors
American Journal of Respiratory Cell and Molecular Biology, 2000Co-Authors: J.c Allo, Patrick Midoux, Michel Monsigny, Pascale Briand, Marc Merten, Evelyne Souil, Joanna Lipecka, Catherine Figarella, Isabelle FajacAbstract:Submucosal gland serous cells are believed to play a major role in the physiopathology of cystic fibrosis (CF) and may represent an important target for CF gene therapy. We have studied the efficiency of reporter gene transfer into immortalized normal (MM-39) and CF (CF-KM4) human airway epithelial gland serous cells using various synthetic vectors: glycosylated Polylysines (glycofectins), polyethylenimine (PEI) (25 and 800 kD), lipofectin, and lipofectAMINE. In both cell lines, a high luciferase activity was achieved with various glycofectins, with PEI 25 kD, and with lipofectAMINE. After three transfections applied daily using alpha-glycosylated Polylysine, 20% of the cells were transfected. At 24 h after CF transmembrane conductance regulator (CFTR) gene transfer into CF-KM4 cells using alpha-glycosylated Polylysine, the immunolocalization of CFTR was analyzed by laser scanning confocal microscopy and the transgenic CFTR was detected by an intense labeling of the plasma membrane. The presence of membrane lectins, i. e., cell surface receptors binding oligosaccharides, was also examined on MM-39 and CF-KM4 cells by assessing the binding and uptake of fluorescein-labeled neoglycoproteins and fluorescein-labeled glycoplexes (glycofectins complexed to plasmid DNA). Among all the neoglycoproteins and glycoplexes tested, those bearing alpha-mannosylated derivatives were most efficiently taken up by both normal and CF gland serous cells. However, alpha-mannosylated Polylysine was quite inefficient for gene transfer, indicating that the efficiency of gene transfer is determined both by the uptake of the complexes and also by their intracellular trafficking. Moreover, our results show that an efficient in vitro gene transfer was achieved in human airway gland serous cells with the same synthetic vectors described to efficiently transfect human airway surface epithelial cells.
-
Sugar-Mediated Uptake of Glycosylated Polylysines and Gene Transfer into Normal and Cystic Fibrosis Airway Epithelial Cells
Human Gene Therapy, 1999Co-Authors: Isabelle Fajac, Michel Monsigny, Pascale Briand, Patrick MidouxAbstract:We have examined the membrane lectin expressed by immortalized normal and cystic fibrosis (CF) airway epithelial cells, using fluorescein-labeled neoglycoproteins; the uptake of plasmid DNA using fluoresceinylated glycoplexes (plasmid/glycosylated Polylysine complexes); and the efficiency of gene transfer when glycosylated Polylysines and glycosylated, partially gluconoylated Polylysines were used as vectors. The most efficient uptake of neoglycoproteins by normal and CF cells was obtained with mannosylated BSA (bovine serum albumin). Similarly, the most efficient uptake of plasmid DNA was obtained with glycoplexes bearing alpha-D-Man residues. Surprisingly, glycoplexes bearing alpha-D-Man residues were poorly efficient for gene transfer into normal and CF cells. The highest luciferase activity was achieved with lactosylated Polylysine- and beta-D-GlcNAc-substituted gluconoylated Polylysine as vectors. Gene transfer efficiency obtained with gluconoylated Polylysine bearing beta-D-GlcNAc residues was similar to that observed with polyethylenimine (PEI; 25 and 800 kDa) and 10-fold higher than that observed with lipofectin and LipofectAMINE. These results suggest that the transfection efficiency with glycoplexes is not determined only by the specificity of the lectin expressed at the cell surface membrane but also by intracellular trafficking of the glycoplexes, which could be mediated by lectins present inside the cells.
Annie Claude Roche - One of the best experts on this subject based on the ideXlab platform.
-
Specific Gene Transfer Based on Biotinylated and Gluconoylated Polylysine/Plasmid Complexes
Drug Delivery, 1997Co-Authors: Patrick Erbacher, Annie Claude Roche, Michel Monsigny, Patrick MidouxAbstract:AbstractComplexes formed between plasmids and Polylysine derivatives bearing recognition signals are the basis of nonviral vehicles suitable for gene delivery into eukaryotic cells by a receptor-mediated endocytosis process. We used an alternative procedure with the aim of achieving noncovalent attachment of recognition signals to plasmids. Biotinylated Polylysine/DNA complexes were made between a plasmid and biotinylated Polylysine by electrostatic interactions, and then the DNA complex was lactosylated via streptavidin bridges in order to target the galactose-specific membrane lectin of human hepatoma (HepG2) cells. HepG2 cells were efficiently transfected in a sugar-dependent manner with a polymer/DNA complex lactosylated with either biotinylated and lactosylated bovine serum albumin or lactosylated streptavidin. In addition, reduction of the number of remaining cationic charges, by partial gluconoylation of biotinylated Polylysine, increased the transfection efficiency of HepG2 cells compared with tha...
-
The reduction of the positive charges of Polylysine by partial gluconoylation increases the transfection efficiency of Polylysine/DNA complexes
Biochimica et Biophysica Acta, 1997Co-Authors: Patrick Erbacher, Annie Claude Roche, Michel Monsigny, Patrick MidouxAbstract:A Polylysine partially substituted with polyhydroxyalkanoyl residues and specially with gluconoyl residues was developed in order to increase the transfection efficiency by decreasing the strength of the electrostatic interactions between the DNA and the cationic polymer. Partially gluconoylated Polylysine/DNA complexes were more easily dissociated in solution and their transfection efficiency in the presence of chloroquine, evaluated with HepG2 cells, a human hepatocarcinoma line, was higher when 43 +/- 4% of the epsilon-amino groups of Polylysine were blocked with gluconoyl residues. Partially gluconoylated Polylysine/plasmid complexes were efficient in transfecting different adherent as well as non-adherent cell lines. Partially gluconoylated Polylysine formed highly soluble (above 100 micrograms/ml in DNA) complexes with DNA plasmids. In addition, partially gluconoylated Polylysine bearing few lactosyl residues increased the transfection efficiency of HepG2 cells which express a galactose-specific membrane lectin.
-
Specific Gene Transfer Based on Biotinylated and Gluconoylated Polylysine/Plasmid Complexes
Drug Delivery, 1997Co-Authors: Patrick Erbacher, Annie Claude Roche, Michel Monsigny, Patrick MidouxAbstract:Complexes formed between plasmids and Polylysine derivatives bearing recognition signals are the basis of nonviral vehicles suitable for gene delivery into eukaryotic cells by a receptor-mediated endocytosis process. We used an alternative procedure with the aim of achieving noncovalent attachment of recognition signals to plasmids. Biotinylated Polylysine/DNA complexes were made between a plasmid and biotinylated Polylysine by electrostatic interactions, and then the DNA complex was lactosylated via streptavidin bridges in order to target the galactose-specific membrane lectin of human hepatoma (HepG2) cells. HepG2 cells were efficiently transfected in a sugar-dependent manner with a polymer/DNA complex lactosylated with either biotinylated and lactosylated bovine serum albumin or lactosylated streptavidin. In addition, reduction of the number of remaining cationic charges, by partial gluconoylation of biotinylated Polylysine, increased the transfection efficiency of HepG2 cells compared with that of lactosylated polymer/DNA complexes. These results indicate that complexes between a plasmid and a partially gluconoylated and biotinylated Polylysine are suitable DNA complexes for the binding of any biotinylated recognition signals for in vitro and ex vivo receptor-mediated gene delivery.
-
Gluconoylated and Glycosylated Polylysines As Vectors for Gene Transfer into Cystic Fibrosis Airway Epithelial Cells
Human Gene Therapy, 1996Co-Authors: Wouter J. W. Kollen, Patrick Midoux, Patrick Erbacher, Alex Yip, Annie Claude Roche, Michel Monsigny, Mary Catherine Glick, Thomas F. ScanlinAbstract:To provide an alternative to viral vectors for the transfer of genes into airway epithelial cells in cystic fibrosis (CF), a novel set of substituted Polylysines were employed. Polylysine was partially neutralized by blocking a number of positively charged residues with gluconoyl groups. In addition, Polylysine was substituted with sugar residues on a specified number of amino groups. Using the gluconoylated Polylysine as vector, the pCMVLuc plasmid gave high expression of the reporter gene luciferase in immortalized CF/T43 cells. The luciferase activity was 75-fold greater in the presence of 100 microM chloroquine. Luciferase gene expression persisted at high levels for up to at least 120 hr following transfection. Glycosylated Polylysines/pCMVLuc complexes were compared to the gluconoylated Polylysine/pCMVLuc complex and beta-Gal-, alpha-Glc-, and Lac-substituted Polylysines gave 320%, 300%, and 290%, respectively, higher expression of the reporter gene luciferase. Luciferase expression ranged from 35 to 2 ng of luciferase per milligram of cell protein in the order: beta-Gal = alpha-Glc = Lac > alpha-Gal = Rha = Man > beta-GalNAc > alpha-GalNAc = alpha-Fuc, suggesting that the transfection efficiency is sugar dependent. Most importantly, in primary cultures of both CF and non-CF airway epithelial cells grown from tracheal tissue explants, lactosylated Polylysine gave uniformly high expression of luciferase. The glycosylated Polylysines provide an attractive nonviral approach for the transfer of genes into airway epithelial cells.
-
Glycosylated Polylysine/DNA complexes: gene transfer efficiency in relation with the size and the sugar substitution level of glycosylated Polylysines and with the plasmid size.
Bioconjugate Chemistry, 1995Co-Authors: Patrick Erbacher, Annie Claude Roche, Michel Monsigny, Patrick MidouxAbstract:A DNA delivery system based on the use of Polylysine substituted with small recognition signals, such as carbohydrate moieties specifically recognized by membrane lectins present in a given cell line, has been developed [Midoux et al. (1993) Nucleic Acids Res. 21, 871-878]. Human hepatoma (HepG2) cells which express a galactose-specific membrane lectin are efficiently transfected in the presence of chloroquine with pSV2Luc plasmid complexed with a lactosylated Polylysine. The optimization of the parameters involved in the formation of DNA/glycosylated Polylysine complexes leads to the following conclusions: a high gene transfer efficiency is reached when (i) DNA/glycosylated Polylysine complexes are completely retarded when subjected to electrophoresis and when (ii) 31 +/- 4% or 40 +/- 8% of the amino groups of a Polylysine having a degree of polymerization (DP) of 190 are substituted with lactosyl or beta-D-galactosyl residues, respectively. In addition, carbohydrate residues bound to Polylysine decrease the electrostatic strength between plasmid DNA and glycosylated Polylysine, suggesting that the strength of the electrostatic interactions between the plasmid and the glycosylated Polylysine plays an important role in the efficiency of the gene expression. The optimal lactosylated Polylysine conjugate (Polylysine DP 190 substituted with 60 lactosyl residues) transfers a 5 kb and a 12 kb plasmid with a similar efficiency.
Max L. Birnstiel - One of the best experts on this subject based on the ideXlab platform.
-
Preparation of adenovirus-Polylysine-DNA complexes.
Current protocols in human genetics, 2001Co-Authors: Matt Cotten, Max L. Birnstiel, Kurt Zatloukal, A Baker, E WagnerAbstract:This unit describes preparation of adenovirus-Polylysine-DNA complexes, which is useful for transfection of DNA into a variety of cell types. A DNA complex is prepared with biotinylated adenovirus and streptavidin-Polylysine, coupled to transferrin, and used to transfect cells. Several support protocols describe methods for adenovirus growth and purification, biotinylation, inactivation with psoralen, and quantitation of the adenovirus particles. Additional support protocols describes preparation of streptavidin-Polylysine and transferrin-Polylysine, necessary for the basic procedure. The DNA used for transfection must be free of lipopolysaccharide (LPS), and two methods for removing LPS are described. A more direct Polylysine-virus linkage that is simple and requires no exotic reagents can be used for transfection. This protocol requires Polylysine-modified adenovirus, prepared as described. An alternate protocol describes transfecting cells with free virus and DNA condensed with a polycation.
-
Current Protocols in Human Genetics - Preparation of Adenovirus‐Polylysine‐DNA Complexes
Current protocols in human genetics, 1996Co-Authors: Matt Cotten, Max L. Birnstiel, Kurt Zatloukal, A Baker, Ernst WagnerAbstract:This unit describes preparation of adenovirus-Polylysine-DNA complexes, which is useful for transfection of DNA into a variety of cell types. A DNA complex is prepared with biotinylated adenovirus and streptavidin-Polylysine, coupled to transferrin, and used to transfect cells. Several support protocols describe methods for adenovirus growth and purification, biotinylation, inactivation with psoralen, and quantitation of the adenovirus particles. Additional support protocols describes preparation of streptavidin-Polylysine and transferrin-Polylysine, necessary for the basic procedure. The DNA used for transfection must be free of lipopolysaccharide (LPS), and two methods for removing LPS are described. A more direct Polylysine-virus linkage that is simple and requires no exotic reagents can be used for transfection. This protocol requires Polylysine-modified adenovirus, prepared as described. An alternate protocol describes transfecting cells with free virus and DNA condensed with a polycation.
-
coupling of adenovirus to transferrin Polylysine dna complexes greatly enhances receptor mediated gene delivery and expression of transfected genes
Proceedings of the National Academy of Sciences of the United States of America, 1992Co-Authors: Ernst Wagner, Matt Cotten, Kurt Zatloukal, Helen Kirlappos, Karl Mechtler, David T Curiel, Max L. BirnstielAbstract:We are developing efficient methods for gene transfer into tissue culture cells. We have previously shown that coupling of a chimeric adenovirus with Polylysine allowed the construction of an adenovirus-Polylysine-reporter-gene complex that transferred the transporter gene with great efficiency into HeLa cells. We have now explored simpler, biochemical means for coupling adenovirus to DNA/Polylysine complexes and show that such complexes yield virtually 100% transfection in tissue culture cell lines. In these methods adenovirus is coupled to Polylysine, either enzymatically through the action of transglutaminase or biochemically by biotinylating adenovirus and streptavidinylating the Polylysine moiety. Combination complexes containing DNA, adenovirus-Polylysine, and transferrin-Polylysine have the capacity to transfer the reporter gene into adenovirus-receptor- and/or transferrin-receptor-rich cells.
-
Influenza virus hemagglutinin HA-2 N-terminal fusogenic peptides augment gene transfer by transferrin-Polylysine-DNA complexes: toward a synthetic virus-like gene-transfer vehicle.
Proceedings of the National Academy of Sciences of the United States of America, 1992Co-Authors: Ernst Wagner, Matt Cotten, Christian Plank, Kurt Zatloukal, Max L. BirnstielAbstract:Complexes containing plasmid DNA, transferrin-Polylysine conjugates, and Polylysine-conjugated peptides derived from the N-terminal sequence of the influenza virus hemagglutinin subunit HA-2 have been used for the transfer of luciferase or beta-galactosidase marker genes to K562 cells, HeLa cells, and BNL CL.2 hepatocytes. These DNA complexes mimic the entry of viruses into cells, as they contain functions for (i) the packaging of the nucleic acid with Polylysine, (ii) the attachment to the cell and receptor-mediated endocytosis with transferrin as a ligand, and (iii) the release from endosomes by using membrane-disrupting influenza peptides. The presence of these influenza peptide conjugates in the DNA complexes renders the complexes active in membrane disruption in a liposome leakage assay and results in a substantial augmentation of the transferrin-Polylysine-mediated gene transfer.
