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Molecular Delivery System for Antisense Oligonucleotides: Enhanced Effectiveness of Antisense Oligonucleotides by HVJ-liposome Mediated Transfer

From the Department of Medicine, Harvard Medical School, and Brigham and Women's Hospital, Boston, Massachusetts

Gary H. Gibbons, MD

From the Department of Medicine, Harvard Medical School, and Brigham and Women's Hospital, Boston, Massachusetts

Masatsugu Horiuchi, MD, PhD

From the Department of Medicine, Harvard Medical School, and Brigham and Women's Hospital, Boston, Massachusetts

Masatoshi Nakajima, PhD

From the Department of Medicine, Harvard Medical School, and Brigham and Women's Hospital, Boston, Massachusetts

Kristin E. Ellison, MD

From the Department of Medicine, Harvard Medical School, and Brigham and Women's Hospital, Boston, Massachusetts

Wendy Lee, MS

From the Department of Medicine, Harvard Medical School, and Brigham and Women's Hospital, Boston, Massachusetts

Yasufumi Kaneda, MD, PhD

Institute for Cellular and Molecular Biology

Toshio Ogihara, MD, PhD

Department of Geriatric Medicine, Osaka University Medical School, Osaka, Japan

Victor J. Dzau, MD

From the Department of Medicine, Harvard Medical School, and Brigham and Women's Hospital, Boston, Massachusetts

Background: The effectiveness of antisense oligodeoxynucleotides for in vitro and in vivo studies is limited by a low efficiency of cellular uptake and instability due to degradation by nucleases. To overcome some of these problems, we recently developed a transfer method that utilizes inactivated Sendai virus (hemagglutinating virus of Japan [HVJ]) complexed with liposomes to deliver antisense oligodeoxynucleotides. In this study, we compared the effectiveness of the HVJ-liposome method versus a cationic liposome method versus passive uptake to deliver antisense oligodeoxynucleotides against basic fibroblast growth factor on angiotensin (Ang) II-induced rat vascular smooth muscle cell growth.

Methods and Results: Twenty to twenty-eight hours after transfection, antisense fibroblast growth factor oligodeoxynucleotides introduced by passive uptake and HVJ-liposome method decreased basal DNA synthesis significantly as compared to the sense, control, and scrambled oligodeoxynucleotides groups; however, 60-68 hours after transfection, only anti sense fibroblast growth factor oligodeoxynucleotides transduced by the HVJ-liposome method resulted in a significant inhibition of DNA synthesis under basal and Ang II-(10^-6M) stimulated conditions. The IC₂₅ of oligodeoxynucleotides assessed by the inhibition of thymidine incorporation was significantly lower using the HVJ-liposome method than those using the other transfer methods. To clarify the mechanisms of cellular uptake of olibodeoxynucleotides with the HVJ-liposome method, we studied the cellular fate of fluo rescein isothiocyanate (FITC)-labeled oligodeoxynucleotides FITC-oligodeoxynucleotides was localized in nuclei at 5 minutes after transfection with the HVJ-liposome method. In contrast, FITC-oligodeoxynucleotides introduced by passive uptake was detected in nonnu clear cellular compartments, possibly endosomes, but not the nuclei. Cellular fluorescence of oligodeoxynucleotides introduced by passive uptake disappeared within 24 hours, while that introduced by the HVJ-liposome method could be observed up to 72 hours.

Conclusion: These results demonstrate that the HVJ-liposome transfer enhanced the effec tiveness of AS-fibroblast growth factor via its specific molecular mechanisms of transfer.

Key Words: gene therapy • fibroblast growth factor • Sendai virus HVJ • antisense technology.

Journal of Cardiovascular Pharmacology and Therapeutics, Vol. 2, No. 3, 213-222 (1997)
DOI: 10.1177/107424849700200308


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