Low levels of gene transfer and a lack of tissue-specific targeting of vectors have limited the therapeutic potential of non-viral gene therapy. This is due to the numerous cellular barriers that hinder nuclear delivery of vectors and the paucity of methods that restrict expression to specific cells types. In non-dividing cells, the nuclear envelope is an especially problematic hurdle to gene transfer. Given that the majority of target tissues are non-dividing in vivo, the nuclear membrane is a major obstacle to therapeutic gene transfer. In this review, the various barriers to gene transfer are discussed. In particular, the role of the nuclear pore complex (NPC) in regulating passage of plasmid vectors during interphase is reviewed. Several methods of modifying plasmid (pDNA) vectors to enhance nuclear import through the NPC are also discussed, including the use of tissue-specific transcription factors to mediate nuclear entry of pDNA in a cell-specific manner.