Nuclear Uptake

Nuclear entry has been described as a formidable barrier to gene delivery [201 -203]. Transport of DNA into this target organelle most likely involves the nuclear pore complex, which has an inner channel size of 9 nm, as determined by electron microscopy [204]. Molecules less than 40-45 kDa diffuse freely through the

NPC, but anything larger must have specific signals, such as nuclear localization sequences to facilitate transport [119].

Several attempts to improve nuclear uptake have been explored. The nuclear localization sequence (NLS) derived from the SV40 large-tumor antigen has been attached to a double-stranded DNA fragment, resulting in enhanced gene expression [205]. The NLSs are thought to enhance nuclear uptake via NPCs (see Ref. 119 for a comprehensive review of NLSs and the biology of NPCs). Adenovirus hexon proteins that mediate translocation of viral genes into the nucleus, through a mechanism separate from NLS-dependent pathways, have also been exploited. PEI/DNA complexes with covalently attached hexon proteins showed 10-fold increase in gene expression in HepG2 cells in vitro [206]. As an alternate approach, a dexamethasone-psoralen conjugate (DR9NP), a steroid derivative, has been complexed with DNA to enhance nuclear uptake [207]. Psoralen binds DNA, and dexamethasone (the steroid) binds its complementary cytoplasmic glucocorti-coid receptor. Subsequent to receptor binding, the complex is transported into the nucleus. This novel method resulted in enhanced gene expression in both dividing and nondividing cells.

Gene delivery into mitotic cells is generally more efficient than in quiescent cells since the nuclear envelope breaks down in the dividing cells, allowing for foreign genes to gain nuclear access [208]. The mitotic activity of the target cells, therefore, may dictate the need for special methods to facilitate nuclear entry. Gene delivery to cells in the lung may involve both quiescent and mitotic cells since lung epithelial cells undergo mitosis periodically.

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