A Regulated, NFκB-Assisted Import of Plasmid DNA into Mammalian Cell Nuclei

• DOI: 10.1006/mthe.2001.0312
• Publication Date: 2003-01-21T13:41:45Z
• Authors: Adi Mesika; Irina Grigoreva; Muriel Zohar; Ziv Reich
• Keywords: Time Factors; Transcription, Genetic; Nuclear Localization Signals; Transfection; Mice; 03 medical and health sciences; Drug Discovery; Genetics; Animals; Humans; Fluorescent Antibody Technique, Indirect; Luciferases; Molecular Biology; Pharmacology; Cell Nucleus; 0303 health sciences; Binding Sites; Tumor Necrosis Factor-alpha; Gene Transfer Techniques; NF-kappa B; Genetic Therapy; Protein Transport; Molecular Medicine; HeLa Cells; Plasmids

The success of synthetic DNA delivery systems in human gene therapy will be enhanced by increasing transfection efficiencies and by providing tighter control over targeting of the DNA into the nucleus. Here, we used DNA vectors that contain repetitive binding sites for the inducible transcription factor NFkappaB, which is transported into the nucleus by the nuclear import machinery. Nuclear entry of the modified vectors was augmented 12-fold and was associated with corresponding increase in gene expression. Depending on their position, the binding sites could also function as transcriptional enhancers, increasing gene expression levels up to an additional 19-fold. Notably, nuclear targeting of the DNA and transgene transcription could both be regulated by exogenous stimulators which modulate the intracellular distribution of NFkappaB. The approach provides a framework for the controlled targeting of constitutive or transcriptionally regulated synthetic vectors into mammalian cell nuclei.