Research Project
6074206
The goal of this proposal is to develop plasmid DNA delivery technology that will efficiently deliver antigen-encoding DNA to antigen presenting cells (APCs) in vivo. This should produce improved transfection of the targeted APCs in vivo and induction of an immune response to the encoded antigen. Plasmid DNA encoding reporter genes or antigens will be condensed, encapsulated, and analyzed for physicochemical properties (e.g. size, mass/charge ratio). The resulting complexes will be mixed with mouse splenocytes, peritoneal macrophages, isolated dendritic cells, and human PBMC preparations in vitro and analyzed for uptake and expression. Flow cytometry will be used to identify the cells that preferentially internalize and express the complexes. If preferential expression is noted in APCs, the ability of anti-CD14 antibodies, LPS, and apoptotic bodies to interfere with this uptake will be tested. Complexes encoding GFP, luciferase or ovalbumin will be injected into mice and analyzed for induction of an antigen specific immune response. If successful, this vehicle could be applied to the formulation of vaccines for infectious disease and cancer. PROPOSED COMMERCIAL APPLICATIONS: Packaging the plasmid DNA with protamine sulfate and anionic lipids is expected to generate a complex that is more resistant to degradation than naked DNA, more stable in storage than attenuated virus, and preferentially internalized by APCs through apoptosis receptors. Such complexes, encoding antigen alone or in combination with genes encoding costimulatory molecules or chemokines, have many potential applications in tumor and infectious disease vaccines. In vivo loading of APCs would be more practical than the ex vivo peptide pulsing of dendritic cells currently in clinical trial.
