(257a) Histone-Mimetic Gold Nanoparticles as Self-Activating & Tailorable Gene Delivery Scaffolds

The field of gene therapy has garnered significant interest over the past two decades as a method for revolutionizing the treatment of various diseases such as Alzheimer's, Parkinson's, and many types of cancer. In recent years, non-viral methods of delivery have received particular attention due to safety concerns and production limitations associated with viral vectors. However, inefficient DNA release is a common cause of ineffective non-viral DNA delivery. A novel solution to this constraint is the development of biomimetic scaffolds capable of regulating DNA accessibility. The presented study involves the design of histone-mimetic gold nanoparticles (HMGNs) as gene therapy packaging materials. Colloidal gold serves as a scaffold for the incorporation of histone H3 tail peptides trimethylated at lysine 4 (H3K4Me3). H3K4Me3 has a high density of positively charged residues that provide a DNA condensation template and impart protection from nuclease degradation. H3K4Me3 is known to be highly enhanced at the transcription start site for essentially all active genes. In addition, recognition of this trimethylated K4-containing peptide sequence by nucleosome remodeling factors has been implicated in mechanisms for chromatin activation. The purpose of the presented work is to assemble and characterize these HMGNs as well as to investigate the influence of HMGN functionalization on DNA binding, protection, and release. To this end, prior to the inclusion of gold particles, the complexation of plasmid DNA by non-trimethylated and trimethylated H3 tail peptides has been studied both independently and in combination with a cationic polymer. The self-assembly of H3K4 and H3K4Me3 with plasmid DNA has been investigated by dynamic light scattering, zeta potential analyses, nuclease assays, and in vitro cell transfection studies. The trafficking of these delivery vehicles through the intracellular network has also been examined. The formation of nanoscale particles that are stable in the presence of serum nucleases was achieved for both H3 peptides formed in combination with PEI. These H3-PEI hybrid vectors were also observed to transfect a substantially higher number of CHO-K1 cells in vitro compared to both complexes that were formed with only the H3 peptides and those formed with only PEI at the same total charge ratio (N:P). The mechanism for this resulting synergistic effect will be studied further. The presented studies are aimed at validating the HMGN approach to gene delivery and will provide the framework for further development of our system.