Large segmental bone defects are a persistent clinical challenge that poses significant economic costs as well as costs to individual health and societal productivity. Limitations in bone grafting emphasize the need for alternative strategies. Accordingly, a myriad of therapeutic approaches have been explored to address this health problem, including the direct delivery of growth factors and delivery of viral growth factor gene therapies. While these approaches have improved healing outcomes, they have not been widely employed clinically, owing at least in part to limited protein stability (direct delivery) and safey concerns (viral gene therapies). Non-viral strategies to induce efficient, cell-mediated production of growth factors would offer a provocative approach to overcome these difficulties. We propose to address these challenges through the development of new, histone-targeted gene transfer scaffolds with tunable DNA binding and controllable gene delivery. The display of histone motifs on nanoparticle scaffolds (e.g. nanogold) will capitalize on our preliminary studies proving that post-translationally modified histone tails promote nuclear accumulation, DNA release, transcription, and enhanced transfection by non-viral vehicles. Additionally, our approach builds on established advantages of nanogold, including imaging potential, biocompatibility, functionalizability, and cell entry capacity. The novel presentation of histone tails on nanogold should mimic the native, multifaceted display and functionality of these sequences on the histone octamer. Hence, these new scaffolds should provide additional important, yet unexplored, benefits for controlling and understanding gene packaging, trafficking, and release through enhanced utilization of native gene transfer and trafficking pathways. Accordingly, the goal of this proposal is to create and optimize multifunctional """"""""designer"""""""" histones to induce efficient gene transfer, and ultimately, enable improved tissue repair for orthopedics and other applications. We will produce nanogold-plasmid DNA (pDNA) assemblies, and will determine whether the multivalent presentation of pDNA-binding residues and histone peptides enhances pDNA-binding stability and improves transfection. We will capitalize on traditional as well as nanogold-specific imaging analyses to elucidate key steps in non-viral gene transfer, and will clearly link enhanced gene transfer efficiency to improved osteogenic potential of growth factors such as bone morphogenetic protein-2 (BMP-2). Finally, we will use murine and rat orthopedic models to test the ability of the BMP-2 gene product to enhance bone formation and increase the speed and efficacy of defect repair. Our approaches will not only elucidate mechanistic details about the trafficking of histone-associated genes, but will also ultimately will be useful as a general biomaterials platform applicable to bone repair, implant functionalization, and tissue engineering.
The purpose of this project is to explore and develop the use of a non-viral targeted gene delivery system to increase the expression of genes including factors that modulate and enhance tissue repair. Our preliminary studies and other published reports indicate that this new technology has the potential to facilitate understanding and control of gene transfer processes, and hence inform fundamental aspects of non-viral carrier design. The proposed studies will demonstrate the capacity of these materials to provide sustained growth factor release and stimulate bone formation, and results generated will provide improved methods for bone defect repair and regeneration with direct applicability to both clinical research and surgical procedures.
|Munsell, Erik V; Ross, Nikki L; Sullivan, Millicent O (2016) Journey to the Center of the Cell: Current Nanocarrier Design Strategies Targeting Biopharmaceuticals to the Cytoplasm and Nucleus. Curr Pharm Des 22:1227-44|
|Ross, Nikki L; Sullivan, Millicent O (2016) Overexpression of caveolin-1 in inflammatory breast cancer cells enables IBC-specific gene delivery and prodrug conversion using histone-targeted polyplexes. Biotechnol Bioeng 113:2686-2697|
|Ross, Nikki L; Sullivan, Millicent O (2015) Importin-4 Regulates Gene Delivery by Enhancing Nuclear Retention and Chromatin Deposition by Polyplexes. Mol Pharm 12:4488-97|