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Identification of human plasma proteins that bind to cationic lipid/DNA complex and analysis of their effects on
Bouna-Moussa Tandia1, Michel Vandenbranden, Ruddy Wattiez
1Laboratory of Structure and Function of Biological Membranes, Center for Structural Biology and Bioinformatics, Université Libre de Bruxelles, Campus Plaine, CP 206/2, B-1050, Brussels, Belgium.
Insights
Plasma proteins and lipoproteins interacting with cationic lipid/DNA complexes significantly impact gene transfer efficiency. Lipoproteins reduce transfection, while fibrinogen enhances it, suggesting cell uptake, not complex breakdown, is key.
Area of Science:
- Biochemistry
- Molecular Biology
- Gene Therapy
Background:
- Cationic lipid/DNA complexes are used for gene therapy but face challenges in intravenous delivery.
- Plasma component interactions with these complexes are poorly understood and limit transfection efficiency.
Purpose of the Study:
- To identify human plasma proteins and lipoproteins that bind to cationic lipid/DNA complexes.
- To investigate the impact of these bound components on gene transfer and expression efficiency in vitro.
- To elucidate the mechanism by which plasma affects complex performance.
Main Methods:
- Isolation of plasma proteins and lipoproteins using sucrose density gradient centrifugation.
- Identification of bound proteins via 2-D gel electrophoresis.
- Assessment of transfection efficiency in lung endothelial cells.
- Quantification of complex uptake using radiolabeled DNA and fluorescence microscopy.
Main Results:
- Protein binding did not cause complex dissociation or DNA degradation.
- Lipoprotein particles significantly reduced the transfection efficiency of the complexes.
- Fibrinogen was found to enhance the transfection process.
- A strong correlation was observed between plasma component effects on transfection and cell uptake efficiency.
Conclusions:
- Complex disruption in plasma is not the primary cause of reduced transfection.
- Coating of complexes by plasma components, particularly lipoproteins, hinders cell uptake, leading to decreased transfection.
- Fibrinogen may play a role in enhancing gene delivery by facilitating complex uptake.
Abstract:
Interaction of cationic lipid/DNA complex with the plasma is a limiting step for the cationic lipid-mediated intravenous gene transfer and expression process. Most of the plasma components that interact with the complex and inhibit its transfection efficiency are still unknown. In the present work, human plasma proteins and lipoproteins that bind to a cationic lipid/DNA complex were isolated on a sucrose density gradient and identified by 2-D gel electrophoresis. Protein binding did not result in complex dissociation or DNA degradation. The effects of several complex-binding plasma components on the transfection efficiency were studied using lung endothelial cells cultured in vitro. Lipoprotein particles caused a drastic loss of the transfection efficiency of the complex. Surprisingly, fibrinogen was found to activate the transfection process. The roles of these complex-binding plasma components on the complex uptake efficiency were quantitatively assessed using radiolabeled plasmid DNA and qualitatively evaluated using fluorescence microscopy. A good correlation was found between the effects of the complex-binding plasma components on the transfection and on cell uptake efficiencies. In contrast to what was generally believed, our data suggest that disruption of the complex does not occur when it is in contact with the plasma and therefore could not be responsible for the loss of transfection activity. Instead, coating of complexes with plasma components seems to be responsible for reduced uptake by cells, which in turn results in reduced transfection.
