Whatever the delivery route used, probably the largest barriers to gene delivery are restricted cytoplasmic diffusion and the nuclear membrane (11,77). DNA particles entering fast dividing cells in culture are less hindered by these problems. Indeed, transfection of synchronized cells showed mitosis to be a key event for efficient transfection (78,79). It is therefore possible that, in general, DNA or DNA complexes that are unable to diffuse through the cytoplasm are dragged along with chromosomes and become incidentally sequestered in the nuclei of daughter cells during telophase. This would explain why most transfected cells appear as doublets (Fig. 9). Despite these observations, there are also quite well-documented cases of linear PEI-based transfection of nondividing cells both in vitro and in vivo. Indeed, in vitro transfection of resting (G1) HeLa cells with linear PEI has been shown to occur (46), albeit with 10-fold less efficiency than dividing ones (G2/M). Lipids or branched PEI were much more dependent on cell division. Moreover, there are a number of studies indicating transfection of mature neurons in vitro and in vivo with linear PEI (62-65,80)
In vivo, even cancer cells can be considered as resting with respect to the lifetime of exogeneous DNA. A vectorial nuclear carrier should therefore be designed. Following the example of some DNA viruses, chemists attempt to divert the endogeneous nuclear import machinery by designing nuclear localization signal peptide (NLS)-bearing vectors (81-86). Unfortunately, cationic vector-DNA complexes are generally
far too large to cross nuclear pores. Assuming the DNA-cati-onic vector complexes are (at least in part) disassembled in the cytoplasm, we have covalently bound a single NLS peptide to one end of a linear DNA fragment (Fig. 10). This hybrid construction was shown to enhance transgene expression up to 1000-fold in HeLa cells (87). Our working hypothesis was that the 3-nm-wide DNA-NLS molecules present in the cytoplasm would initially be translocated through a nuclear pore by the nuclear import machinery. As DNA would enter the nucleus, it would be condensed into a chromatinlike structure, which would provide the mechanism for threading the remaining wormlike molecule through the pore. Unfortunately, large enhancement factors were only observed for cell lines. Resting primary cells remained refractory, which may indicate that the large intracellular motions that occur during mitosis are required for intracellular trafficking of the DNA-NLS as well.
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