Reconstituted influenza virosomes (virus membrane envelopes) have been used previously to deliver pDNA (plasmid DNA) bound to their external surface to a variety of target cells. Although high transfection efficiencies can be obtained with these complexes in vitro, the virosome-associated DNA is readily accessible to nucleases and could therefore be prone to rapid degradation under in vivo conditions. In the present study, we show a new method for the production of DNA–virosomes resulting in complete protection of the DNA from nucleases. This method relies on the use of the short-chain phospholipid DCPC (dicaproylphosphatidylcholine) for solubilization of the viral membrane. The solubilized viral membrane components are mixed with pDNA and cationic lipid. Reconstitution of the viral envelopes and simultaneous encapsulation of pDNA is achieved by removal of the DCPC from the mixture through dialysis. Analysis by linear sucrose density-gradient centrifugation revealed that protein, phospholipid and pDNA physically associated to particles, which appeared as vesicles with spike proteins inserted in their membranes when analysed by electron microscopy. The DNA–virosomes retained the membrane fusion properties of the native influenza virus. The virosome-associated pDNA was completely protected from degradation by nucleases, providing evidence for the DNA being highly condensed and encapsulated in the lumen of the virosomes. DNA–virosomes, containing reporter gene constructs, transfected a variety of cell lines, with efficiencies approaching 90%. Transfection was completely dependent on the fusogenic properties of the viral spike protein haemagglutinin. Thus, DNA–virosomes prepared by the new procedure are highly efficient vehicles for DNA delivery, offering the advantage of complete DNA protection, which is especially important for future in vivo applications.
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Research Article|
June 13 2007
Cellular gene transfer mediated by influenza virosomes with encapsulated plasmid DNA
Jørgen de Jonge;
Jørgen de Jonge
1
*Department of Medical Microbiology, Molecular Virology Section, University Medical Center Groningen and University of Groningen, Groningen, The Netherlands
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Johanna M. Leenhouts;
Johanna M. Leenhouts
†Department of Biochemistry and Molecular Biology, University of British Columbia, Vancouver, BC, Canada
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Marijke Holtrop;
Marijke Holtrop
*Department of Medical Microbiology, Molecular Virology Section, University Medical Center Groningen and University of Groningen, Groningen, The Netherlands
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Pieter Schoen;
Pieter Schoen
1
*Department of Medical Microbiology, Molecular Virology Section, University Medical Center Groningen and University of Groningen, Groningen, The Netherlands
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Peter Scherrer;
Peter Scherrer
2
†Department of Biochemistry and Molecular Biology, University of British Columbia, Vancouver, BC, Canada
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Pieter R. Cullis;
Pieter R. Cullis
†Department of Biochemistry and Molecular Biology, University of British Columbia, Vancouver, BC, Canada
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Jan Wilschut;
Jan Wilschut
*Department of Medical Microbiology, Molecular Virology Section, University Medical Center Groningen and University of Groningen, Groningen, The Netherlands
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Anke Huckriede
Anke Huckriede
3
*Department of Medical Microbiology, Molecular Virology Section, University Medical Center Groningen and University of Groningen, Groningen, The Netherlands
3To whom correspondence should be addressed (email a.l.w.huckriede@med.umcg.nl).
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Publisher: Portland Press Ltd
Received:
November 24 2006
Revision Received:
March 02 2007
Accepted:
March 13 2007
Accepted Manuscript online:
March 13 2007
Online ISSN: 1470-8728
Print ISSN: 0264-6021
© The Authors Journal compilation © 2007 Biochemical Society
2007
Biochem J (2007) 405 (1): 41–49.
Article history
Received:
November 24 2006
Revision Received:
March 02 2007
Accepted:
March 13 2007
Accepted Manuscript online:
March 13 2007
Citation
Jørgen de Jonge, Johanna M. Leenhouts, Marijke Holtrop, Pieter Schoen, Peter Scherrer, Pieter R. Cullis, Jan Wilschut, Anke Huckriede; Cellular gene transfer mediated by influenza virosomes with encapsulated plasmid DNA. Biochem J 1 July 2007; 405 (1): 41–49. doi: https://doi.org/10.1042/BJ20061756
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