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A reliable new procedure is described for the reconstitution of Sendai viral envelopes suitable for gene transfer. Both fusion and hemagglutinin-neuraminidase glycoproteins were extracted from purified Sendai virus and reconstituted together with DNA in the presence of cholesterol:sphingomyelin:phosphatidylcholine:phosphatidylethanolamin e (Chol:SM:PC:PE) in a molar ratio of 3.5:3.5:2:1. Before reconstitution, the DNA to be transferred was condensed by pretreatment with polylysine. Exogenous lipid addition and the DNA-condensation step were essential for maximal size as well as for fusogenic activity of the resulting virosomes, the analysis of which revealed (1) the absence of any genomic material originating from Sendai virus, (2) the presence of fusogenic spikes in a functional orientation, (3) the encapsulation of reporter genes, and (4) high-transfer activity for plasmids carrying the green fluorescent protein (GFP) gene and double-stranded nucleotides into different mammalian cells. Transfer rates were up to 10-fold higher than those obtained with different cationic lipids. Gene delivery by means of our lipid-enriched Sendai virosomes extends the known gene transfer strategies, including those based on Sendai virus previously published.