The design of synthetic vectors for gene transfer has evolved, to some extent, through comparisons with the mechanisms used by natural gene transfer vectors (viruses). Many of these mechanisms, including affinity for a cell surface receptor, enhanced escape from acidic endosomes, condensation of DNA and nuclear targeting, have been or are currently being engineered into synthetic vectors. It follows that further descriptions of viral infection mechanisms will provide additional clues for creating more efficient synthetic vectors. Toward this end, we are investigating molecular mechanisms used by adenoviral vectors during intracellular trafficking of the viral genome to the nucleus of the target cell. Part of the efficiency with which adenoviral vectors infect target cells comes from the fact that approximately 80% of viral capsids and genome achieve nuclear localization within 60 min after infection. With a diameter of 80 nm, the adenovirus capsid is too large to diffuse through the cytosol leading to the hypothesis that the adenovirus capsid requires an active transport mechanism to move efficiently to the nucleus. Our studies show that nuclear localization of adenoviral capsids requires the presence of intact microtubules and can be reversibly inhibited by the microtubule depolymerizing agent, nocodozole. Adenovirus movement to the nucleus is also inhibited in the presence of function blocking antibodies to the microtubule-associated molecular motor, cytoplasmic dynein. When both microtubules and dynein are available, adenovirus achieves nuclear localization, at times moving through the cytoplasm with a velocity of 2.2 microns per second. Extrapolating these results to synthetic vector systems, it is likely that incorporation of a mechanism for utilizing microtubule-dependent translocation into the design of synthetic vectors has potential to increase the efficiency of gene transfer.
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