Collins Elizabeth, Birchall James C, Williams Julie L, Gumbleton Mark
Welsh School of Pharmacy, Cardiff University, Cardiff, UK.
J Gene Med. 2007 Apr;9(4):265-74. doi: 10.1002/jgm.1015.
Non-viral gene delivery vectors are multi-component systems reflecting various functionalities required for effective cell transfection, including DNA condensation, promotion of cell membrane interactions and provision for subcellular targeting through endosomal escape and/or nuclear delivery. Elements mediating these functions will clearly display inter-dependency. In this study we sought to explore the relationship within non-viral vectors of condensation and nuclear localisation.
Binary, tertiary and quaternary vectors were prepared with combinations of pDNA, DOTAP lipid, the polycation peptide protamine and either SV40 nuclear localisation sequence peptide ('SV40 NLS') or a one amino acid substituted mutant of SV40 NLS ('mutant sequence'). The efficiency of pDNA condensation was determined by gel electrophoresis and quantitative fluorescence spectroscopy. Transfection efficiency was examined in mammalian cells in vitro using standard methods, by electroporation to bypass the plasma membrane barrier and in cells arrested in G0/G1 cell cycle phase to examine the effect of cell division and nuclear membrane disruption.
Small NLS peptide sequences, despite possessing a significant proportion of basic amino acids, display minimal pDNA-condensing ability when compared to larger polycations such as protamine. In standard in vitro cell adherent transfection studies the predominant elements affording enhanced gene expression were effective pDNA condensation and lipid enhancement of cell membrane interactions. These features conversely hinder efficient gene expression in cells that have undergone electroporation. The benefit of SV40 NLS was only apparent when used in non-dividing cell populations.
Whilst effective levels of non-viral-mediated gene expression generally rely on efficient condensation of pDNA and enhanced interactions with cellular membranes, non-covalently associated NLS within a multi-component non-viral gene vector appears to contribute benefit in sustaining gene expression in non-dividing cells.
非病毒基因递送载体是多组分系统，反映了有效细胞转染所需的各种功能，包括DNA凝聚、促进细胞膜相互作用以及通过内体逃逸和/或核递送实现亚细胞靶向。介导这些功能的元件显然会表现出相互依赖性。在本研究中，我们试图探索非病毒载体中凝聚与核定位之间的关系。
用pDNA、DOTAP脂质、聚阳离子肽鱼精蛋白以及SV40核定位序列肽（“SV40 NLS”）或SV40 NLS的单氨基酸取代突变体（“突变序列”）组合制备二元、三元和四元载体。通过凝胶电泳和定量荧光光谱法测定pDNA凝聚效率。使用标准方法在体外哺乳动物细胞中检测转染效率，通过电穿孔绕过质膜屏障，并在G0/G1细胞周期阶段停滞的细胞中检测细胞分裂和核膜破坏的影响。
尽管小的NLS肽序列含有相当比例的碱性氨基酸，但与较大的聚阳离子如鱼精蛋白相比，其pDNA凝聚能力最小。在标准的体外细胞贴壁转染研究中，增强基因表达的主要因素是有效的pDNA凝聚和脂质增强的细胞膜相互作用。相反，这些特征会阻碍经电穿孔处理的细胞中的有效基因表达。SV40 NLS的益处仅在非分裂细胞群体中使用时才明显。
虽然非病毒介导的基因表达的有效水平通常依赖于pDNA的有效凝聚和与细胞膜的增强相互作用，但多组分非病毒基因载体中与非共价结合的NLS似乎有助于在非分裂细胞中维持基因表达。
