反义寡核苷酸的分子递送系统：通过HVJ-脂质体介导的转移增强反义寡核苷酸的有效性

Morishita R, Gibbons GH, Horiuchi M, Nakajima M, Ellison KE, Lee W, Kaneda Y, Ogihara T, Dzau VJ

Department of Medicine, Harvard Medical School, Boston, Massachusetts, USA

J Cardiovasc Pharmacol Ther. 1997 Jul;2(3):213-222. doi: 10.1177/107424849700200308.

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 antisense fibroblast growth factor oligodeoxynucleotides transduced by the HVJ-liposome method resulted in a significant inhibition of DNA synthesis under basal and Ang II-(10(-;6)M) stimulated conditions. The IC(25) 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 oligodeoxynucleotides with the HVJ-liposome method, we studied the cellular fate of fluorescein 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 nonnuclear 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 effectiveness of AS-fibroblast growth factor via its specific molecular mechanisms of transfer.

背景

反义寡脱氧核苷酸在体外和体内研究中的有效性受到细胞摄取效率低以及核酸酶降解导致的不稳定性的限制。为了克服其中一些问题，我们最近开发了一种转移方法，该方法利用与脂质体复合的灭活仙台病毒（日本血凝病毒[HVJ]）来递送反义寡脱氧核苷酸。在本研究中，我们比较了HVJ-脂质体方法、阳离子脂质体方法和被动摄取方法在递送针对碱性成纤维细胞生长因子的反义寡脱氧核苷酸以抑制血管紧张素（Ang）II诱导的大鼠血管平滑肌细胞生长方面的有效性。

方法与结果

转染后20至28小时，与正义、对照和乱序寡核苷酸组相比，通过被动摄取和HVJ-脂质体方法引入的反义成纤维细胞生长因子寡脱氧核苷酸显著降低了基础DNA合成；然而，转染后60至68小时，仅通过HVJ-脂质体方法转导的反义成纤维细胞生长因子寡脱氧核苷酸在基础和Ang II-（10⁻⁶M）刺激条件下导致DNA合成的显著抑制。通过抑制胸苷掺入评估的寡脱氧核苷酸的IC₂₅，使用HVJ-脂质体方法明显低于使用其他转移方法。为了阐明HVJ-脂质体方法摄取寡脱氧核苷酸的细胞机制，我们研究了异硫氰酸荧光素（FITC）标记的寡脱氧核苷酸的细胞命运。用HVJ-脂质体方法转染后5分钟，FITC-寡脱氧核苷酸定位于细胞核中。相比之下，通过被动摄取引入的FITC-寡脱氧核苷酸在非核细胞区室（可能是内体）中被检测到，但不在细胞核中。通过被动摄取引入的寡脱氧核苷酸的细胞荧光在24小时内消失，而通过HVJ-脂质体方法引入的细胞荧光可持续观察到72小时。