(731d) Histone Polyplexes Utilize Caveolar Uptake and Traffic Through the Golgi and ER During Gene Transfer

Improved control over the subcellular trafficking of polyplexes has been widely identified as a key hurdle towards improving their activity in the in vivo setting, where gene transfer inefficiencies cannot be easily offset by increasing the concentration of the delivered vehicle.  It has become increasingly apparent that the endocytic uptake route for polyplexes is a fundamental determinant of intracellular trafficking and activity.  Our recent findings suggest that caveolar uptake may be involved in transfection by polyplexes comprised of pDNA, a histone H3 sequence involved in nuclear localization and chromatin activation, and branched poly(ethylenimine) (PEI).  We have found that polyplexes containing ~90% (w/w) H3 tail peptides and ~10% PEI produced significantly more robust transfection and lower cytotoxicity than PEI-pDNA polyplexes.  We hypothesized that the improved activities of the hybrid H3/PEI-pDNA polyplexes as compared with PEI-pDNA polyplexes were caused by their altered intracellular trafficking and enhanced nuclear delivery.  H3/PEI-pDNA polyplexes exhibited slower uptake and a reduced dependence on endocytic pathways that trafficked to the lysosome in comparison with PEI-pDNA polyplexes.  Furthermore, treatment with the macrolide antibiotic bafilomycin A1 significantly reduced H3/PEI-pDNA transfection, but had less substantial effects on PEI-pDNA transfection.  The V-type ATPase binding partners of bafilomycin A1 colocalize with caveolin-1, and bafilomycin A1 alters retrograde trafficking from the Golgi complex.  Thus, these results may suggest that trafficking through caveolae and the Golgi complex represents a productive pathway that enhances H3/PEI-pDNA polyplex delivery to the nucleus.