FUSIGENIC LIPOSOME-MEDIATED DNA TRANSFER INTO CARDIAC MYOCYTES

Current methods of gene transfer into cultured cardiac myocytes have s erious limitations, including low efficiency, toxicity or constraints on DNA insert size, The present study examined the effectiveness of he magglutinating virus of Japan (HVJ) in promoting liposome-mediated DNA transfer into cultured neonatal rat cardiac myocytes, Fluorescein iso thiocyanate-labeled oligonucleotides (F-ODN) or plasmid expression vec tors encoding SV40 large T antigen (pActSVT) and beta-galactosidase (p Act beta-gal) were complexed with liposomes and the viral protein coat of HVJ. Plasmid vectors were complexed with the nuclear localizing pr otein HMG-1 prior to HVJ-liposome encapsulation, Neonatal myocytes wer e transfected by incubation with HVJ-liposome/DNA complexes on culture day 3 or 7. Using F-ODN. we were able to demonstrate significant upta ke of DNA (transfection efficiencies of 80-90%) 1 h after transfection that persisted for 1 week in culture, Interestingly, F-ODN were conce ntrated in the myocyte nuclei for the first 4 days after transfection, Immunohistochemistry showed that 25-30% of myocytes transfected with either pActSVT or pAct beta-Gal expressed plasmid-encoded protein at 7 2 h whether they were transfected at day 3 or day 7 of culture, while cells transfected with blank vectors did not, Quantitative beta-galact osidase assays confirmed that the use of HVJ significantly enhanced li posome-mediated transfection. Cell toxicity was not apparent, Gene tra nsfer via intracoronary injection also demonstrated the capacity of HV J to mediate transfection of rabbit cardiac myocytes in vivo, with F-O DN-dependent fluorescence persisting for up to 1 week. We conclude tha t HVJ/liposome-mediated transfer is efficient for the transfection of both oligonucleotides and plasmids into cardiac myocytes both in vitro and in vivo, and may provide a new tool for the investigation of card iac myocyte biology and disease. (C) 1996 Academic Press Limited.