Lewis J G, Lin K Y, Kothavale A, Flanagan W M, Matteucci M D, DePrince R B, Mook R A, Hendren R W, Wagner R W
Gilead Sciences, Foster City, CA 94404, USA.
Proc Natl Acad Sci U S A. 1996 Apr 16;93(8):3176-81. doi: 10.1073/pnas.93.8.3176.
Development of antisense technology has focused in part on creating improved methods for delivering oligodeoxynucleotides (ODNs) to cells. In this report, we describe a cationic lipid that, when formulated with the fusogenic lipid dioleoylphosphatidyliethanolamine, greatly improves the cellular uptake properties of antisense ODNs, as well as plasmid DNA. This lipid formulation, termed GS 2888 cytofectin, (i) efficiently transfects ODNs and plasmids into many cell types in the presence or absence of 10% serum in the medium, (ii) uses a 4- to 10-fold lower concentration of the agent as compared to the commercially available Lipofectin liposome, and (iii) is > or = 20-fold more effective at eliciting antisense effects in the presence of serum when compared to Lipofectin. Here we show antisense effects using GS 2888 cytofectin together with C-5 propynyl pyrimidine phosphorothioate ODNs in which we achieve inhibition of gene expression using low nanomolar concentrations of ODN. This agent expands the utility of antisense ODNs for their use in understanding gene function and offers the potential for its use in DNA delivery applications in vivo.
反义技术的发展部分集中在开发将寡脱氧核苷酸(ODN)递送至细胞的改进方法。在本报告中,我们描述了一种阳离子脂质,当与促融合脂质二油酰磷脂酰乙醇胺一起配制时,能极大地改善反义ODN以及质粒DNA的细胞摄取特性。这种脂质制剂称为GS 2888细胞转染剂,(i)在培养基中存在或不存在10%血清的情况下,能有效地将ODN和质粒转染到多种细胞类型中,(ii)与市售的Lipofectin脂质体相比,使用的该制剂浓度低4至10倍,(iii)与Lipofectin相比,在有血清存在时引发反义效应的效果高20倍或更高。在此我们展示了使用GS 2888细胞转染剂与C-5炔丙基嘧啶硫代磷酸酯ODN一起产生的反义效应,其中我们使用低纳摩尔浓度的ODN实现了基因表达的抑制。这种制剂扩展了反义ODN在理解基因功能方面的用途,并为其在体内DNA递送应用中的使用提供了潜力。