Gene nuclear targeting

Chan CK, Jans DA. Using nuclear targeting signals to enhance non-viral gene transfer. Immunol Cell Biol 2002 80(2) 119-130. 

In mammalian cells the El ubiquitin-activating enzyme exists in two isoforms, Ela (110 kDa) and Elb (117 kDa), which are derived from a single gene and mRNA (Cook and Chock 1992, Handley Gearhart et al. 1994). The isoform Ela is predominantly found in the nucleus and has been shown to harbor a functional nuclear localization sequence (NLS) required for nuclear targeting and phosphorylation. In contrast, Elb lacks the NLS, is not phos-phorylated and localized in the cytoplasm (Handley-Gearhart et al. 1994 Stephen et al. 1997). Phosphorylation of Ela was demonstrated to occur in a cell cycle-dependent manner, being maximal in G2 phase (Stephen et al. 

While isolation of a specific inhibitor will be necessary to assess the definitive role of the cytosolic nuclease in the low transfection efficiency in vivo, circumstantial evidence suggests that the metabolic instability of plasmid DNA represents one of the cellular barriers to gene transfer. Microinjection of DNA complexes with PEI has augmented the transfection efficiency (Pollard et al., 1998). Although the stability of the PEI-complexed DNA has not been determined in vivo, it has been demonstrated that the nuclease resistance of plasmid DNA is dramatically increased upon complex formation in vitro (Cappaccioli et al., 1993 Chiou et al., 1994 Thierry et al., 1997). Therefore, it is conceivable that faster diffusional mobility and decreased nuclease susceptibility jointly lead to the enhanced nuclear targeting efficiency of the PEI-condensed plasmid DNA. 

As with vitamin A, mast of the effects of vitamin D involve a nuclear receptor. The vitamin D receptor is a member of the steroid/thyroid hormone superfamily of receptors. When I a,2S-OHD binds to its receptor, the complex forms a hetcrodimer with an unoccupied RXR. This heterodimer subsequently binds to the regulatory regions on specific genes in target tissue. These regions are called vitamin D response elements (VDREs). The binding to VDREs can increase or decrease expression of genes. The proteins thus made carry out the functions of vitamin D. 

For nonviral systems to be effective vectors, the polymer must not only condense and deliver DNA intact to the target site but must also enable nucleic acid to be transported through the cell membrane, and be translocated from the cytoplasm to the nucleus. Many researchers have now investigated DNA complexed by electrostatic interactions to cationic polymers as a method by which the therapeutic gene can be transported. In most cases these cationic polymers form condensed complexes with DNA that both contract the nucleic acid to facilitate cellular uptake, and which protect it from serum and cytosolic nuclease degradation. Mechanisms of DNA condensation, cellular uptake and transport to the nucleus, as well as strategies to improve toxicity, transfection potential and nuclear targeting of polycation mediated dehvery systems are discussed below. 

Lentiviruses are promising vectors for gene delivery in vivo with many in precli-nical development for gene therapy (115). The mechanism of nuclear targeting... 

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