Nucleotide exchange in genomic DNA of rat hepatocytes using RNA/DNA oligonucleotides - Targeted delivery of liposomes and polyethyleneimine to the asialoglycoprotein receptor
Nucleotide exchange in genomic DNA of rat hepatocytes using RNA/DNA oligonucleotides - Targeted delivery of liposomes and polyethyleneimine to the asialoglycoprotein receptor
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DOI：
10.1074/jbc.274.15.10163
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发表时间：
1999-04-09
影响因子：
4.8
通讯作者：
Steer, CJ
中科院分区：
文献类型：
--
作者：
Bandyopadhyay, P;Ma, XM;Steer, CJ
嵌合RNA/DNA寡核苷酸已显示促进基因组DNA中的单核苷酸交换。设计嵌合分子以在大鼠因子IX基因的Ser（365）位置处引入A至C核苷酸转换。将寡核苷酸包封在含有半乳糖苷或与乳糖基化聚乙烯亚胺复合的带正电荷、中性电荷和带负电荷的脂质体中。评价制剂的稳定性和通过脱唾液酸糖蛋白受体靶向肝细胞的效率。物理表征和电子显微镜显示，寡核苷酸被有效地包封在脂质体中，阳性和阴性制剂保持稳定至少1个月。每种制剂在分离的大鼠肝细胞中的转染效率接近100%。然而，负脂质体和25-kDa乳糖基化聚乙烯亚胺提供了最强烈的核荧光与荧光素标记的寡核苷酸。乳糖基化聚乙烯亚胺和三种不同的脂质体制剂导致19- 24%的A至C转化效率。此外，乳糖基化聚乙烯亚胺在将质粒DNA转染到分离的肝细胞中也是高度有效的。结果表明，脂质体和聚乙烯亚胺制剂制备简单且稳定，并给出可靠、可重现的结果。它们为肝细胞提供了有效的递送系统，用于通过嵌合RNA/DNA寡核苷酸引入或修复遗传突变。
Chimeric RNA/DNA oligonucleotides have been shown to promote single nucleotide exchange in genomic DNA. A chimeric molecule was designed to introduce an A to C nucleotide conversion at the Ser(365) position of the rat factor IX gene. The oligonucleotides were encapsulated in positive, neutral, and negatively charged liposomes containing galactocerebroside or complexed with lactosylated polyethyleneimine. The formulations were evaluated for stability and efficiency in targeting hepatocytes via the asialoglycoprotein receptor. Physical characterization and electron microscopy revealed that the oligonucleotides were efficiently encapsulated within the liposomes, with the positive and negative formulations remaining stable for at least 1 month. Transfection efficiencies in isolated rat hepatocytes approached 100% with each of the formulations. However, the negative liposomes and 25-kDa lactosylated polyethyleneimine provided the most intense nuclear fluorescence with the fluorescein-labeled oligonucleotides. The lactosylated polyethyleneimine and the three different liposomal formulations resulted in A to C conversion efficiencies of 19-24%. In addition, lactosylated polyethyleneimine was also highly effective in transfecting plasmid DNA into isolated hepatocytes. The results suggest that both the liposomal and polyethyleneimine formulations are simple to prepare and stable and give reliable, reproducible results. They provide efficient delivery systems to hepatocytes for the introduction or repair of genetic mutations by the chimeric RNA/DNA oligonucleotides.