The overall objective of this proposal is to develop biocompatible, biodegradable and self- assembling copolymer/DNA micelles to achieve efficient liver-targeted gene delivery. Liver is a critically important target for gene medicine applications because of the access of the transgene product to systemic circulation, and because it is the site of many metabolic genetic disorders, viral infection and malignancies. At present, the full potential of liver-targeted gene transfer is hindered by a lack of safe and efficient gene carriers. Polymeric micelles with a DNA/polycation complex core and a hydrophilic corona represent a promising carrier for liver-targeted gene delivery. These DNA-encapsulating micelles exhibit several desirable features that favor liver- targeted gene delivery, such as small size (80-100 nm), reduced interaction with biological components, prolonged circulation in blood, and low toxicity. Novel PEG-b-PPA/DNA micelles exhibit high DNA binding capacity, in vitro transfection efficiency, low cytotoxicity and good tissue biocompatibility. In this study, we will (1) synthesize and characterize a mini-library of PEG-b-PPA/DNA micelles with improved complex stability, colloidal stability, intracellular DNA release, endosomal escape, and hepatocyte-targeting ability;(2) characterize the transfection efficiency and cytotoxicity in rat primary hepatocytes, Kupffer cells and normal rat cholangiocytes for this series of structurally distinct copolymers/micelles, and correlate with micelle structures;(3) optimize the administration parameters to achieve efficient liver gene expression for both a non-secretory protein (luciferase) and a secretory protein (interferon-a2b). This will demonstrate the broad utility of this delivery strategy for expression of proteins intended for systemic distribution and for localized liver-specific diseases. Mechanistic studies will be performed in vitro and in vivo to understand the extracellular and intracellular transport of the micelles. This study integrates expertise in polymer chemistry, biomaterial design, molecular/cell biology and clinical sciences. It represents a systematic design and optimization of gene delivery strategies at multiple levels. This study will lay the foundation for future clinical application of polymeric micelles for liver-targeted gene therapy. Project Narrative The overall objective of this proposal is to develop safe and efficient non-viral nanoparticle carriers for gene delivery to the liver. Should this proposed study be successful, these new gene carriers will find wide clinical applications for treating inherited and metabolic liver diseases, liver cancer, viral hepatitis, and systemic diseases, like hemophilia A and B.
