(352b) Intracellular Trafficking and Activity of Histone-Mimetic Gene Delivery Vehicles

Effective gene transfer strategies would fundamentally alter healthcare, and depend upon our ability to create materials that can chaperone DNA transport and release within a diverse biological environment.  A critical problem in non-viral gene therapy is how to trigger nucleus-specific DNA release:  current electrostatic DNA packaging techniques condense DNA to prohibit DNase access, but are believed to inhibit transcription in the process.  We have created a new class of DNA packaging materials that are designed to promote efficient transcription within the nucleus by interacting with histone effector proteins.  These materials contain peptides that are comprised of histone tail sequences known to signal nuclear import and transcriptional activation.  In this work, we have explored the activity of polyplexes containing trimethylated H3 histone tail peptides (H3K4Me₃) that are known to interact with two nucleus-specific effector complexes that initiate transcriptional activation.  We demonstrate that these materials initiate gene expression more rapidly than traditionally packaged polyplexes or polyplexes made from non-methylated H3 histone tail peptides when introduced directly into the nucleus.  Furthermore, we have developed a simple mass action kinetic model that demonstrates that the enhanced activity of the H3K4Me₃-containing polyplexes results from their more rapid nuclear unpackaging/activation.  We have used co-immunoprecipitation to show that the H3K4Me₃–containing polyplexes interact with elements of the transcriptional machinery, presumably as a result of their specific interactions with resident nuclear proteins.  Finally, when the H3K4Me₃ peptides are combined with traditional gene transfer polycations such as polyethylenimine (PEI), the hybrid polyplexes promote higher levels of gene transfer into cultured cells than PEI alone, suggesting that nuclear unpackaging and activation represent significant barriers to effective gene transfer.