Reconstituted fusion liposomes for gene transfer in vitro and in vivo

Abstract

Using UV-inactivated whole HVJ (Hemagglutinating Virus of Japan; Sendai virus), we have developed HVJ-liposomes that are efficient in vitro and in vivo gene delivery vehicles based on cell fusion properties of the Sendai virus. However, all the proteins and the genome of HVJ remain within the HVJ-liposomes, although replication of the viral genome is severely impaired by prior UV-irradiation of HVJ particles. To construct more complete synthetic vehicles, we developed reconstituted fusion liposomes. Fusion proteins F1 and HN of HVJ were extracted by mild lysis of the viral particles and purified by ion-exchange column chromatography. Purified viral fusion proteins were inserted into liposome membranes by detergent-solubilization and dialysis to construct the reconstituted fusion particles. These particles retained fusion activity during more than 4 weeks. DNA-loaded liposomes, which were prepared by vortexing-sonication, were fused with the reconstituted fusion particles to deliver DNA to cells. Using the reconstituted vehicle, fluorescent isothiocyanate (FITC)-labeled oligonucleotides were introduced into 100% of the nuclei of target human amniotic FL cells. In addition, luciferase gene expression upon transfection of human 293 cells with reconstituted fusion liposomes was almost the same as with standard HVJ-liposomes. On the other hand, the LacZ gene was introduced into mouse skeletal muscle by the new vector, and 40 to 50% of the muscle fibers showed LacZ gene expression.