Successful gene therapy mandates the use of delivery vectors. Virus-based delivery systems may entail adverse immune responses in humans. Because of this safety-related concern, synthetic non-viral vector systems are actively being pursued at the present time. Approaches involvingpolymers and phospholipids are commonly referred to as polyplex and lipoplex, respectively. In this study, we explore further the potential utility of nanogel systems we have recently developed for a polyplex gene delivery system. Nanogels are synthesized by means of radical polymerization of inverse water-in-oil micelles. Since the nanoparticles are formed in aqueous droplets of approximately 50 nanometers, it is an ideal system for condensing and entrapping genetic materials. While this is a novel approach, we can alternatively allow pre-made positively charged nanogels to form complexes with plasmid DNA. In either approach, release from the nanogel matrix and translocation of nucleic acids to cytoplasm from the initial location in the cell (i.e., endosomes) must be incorporated into the polyplex design. We plan to achieve these goals by introducing acid-labile crosslinkers and weak bases to the nanogel matrix, respectively.Both strategies are based on the acidity of endosome, approximately 5. Cell-based luciferase expression systems will be used in assessing the performance of formulations. Genetic materials to be studied include small antisense oligonucleotides of about 20 nucleotides, double stranded small interfering RNAs (siRNA), and a large plasmid DNA consisting of 5,900 base pairs. Formulations selected from in vitro cell experiments will be subject to pharmacokinetic studies of gene expression in mice using a real-time bioluminescence imaging system. This is in collaboration with the Samulski laboratory. Pharmcodynamic studies involving antisense and siRNA will be tested in animal models of human prostate cancer, a human breast cancer model, and intimal hyperplasia. These studies are in collaboration with the Kole, Juliano, and Sullenger laboratories, respectively, Successful outcome of the present studies should have important implications for the future of human gene therapy.