Cellular gene transfer mediated by influenza virosomes with encapsulated plasmid DNA

Jorgen de Jonge , Johanna M. Leenhouts , Marijke Holtrop , Pieter Schoen , Peter Scherrer , Pieter R. Cullis , Jan Wilschut , Anke Huckriede (1)

1 Medical Microbiology

Reconstituted influenza virus membrane envelopes (virosomes) have been used previously to deliver plasmid DNA (pDNA) bound to their external surface to a variety of target cells. While high transfection efficiencies can be obtained with these complexes in vitro, the virosome-associated DNA is readily accessible for nucleases and could therefore be prone to rapid degradation under in vivo conditions. Here we present 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 dicaproylphosphatidylcholine (DCPC) 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 analyzed 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 added 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 hemagglutinin. 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.

Mots clés: Life Sciences

Jorgen de Jonge, Johanna M. Leenhouts, Marijke Holtrop, Pieter Schoen, Peter Scherrer, et al.. Cellular gene transfer mediated by influenza virosomes with encapsulated plasmid DNA. Biochemical Journal, 2007, 405 (1), pp.41-49. ⟨10.1042/BJ20061756⟩. ⟨hal-00478703⟩