We will establish simple and convenient methods to knockout specific gene expression in animals. Antisense oligonucleotides (ASO) and antisense genes (ASG) can selectively suppress gene function by interacting with the message of interest to eliminate a specific protein. This approach is currently unfeasible in animals because of pharmacokinetic and transport (both into the cytoplasm and into the nucleus) considerations. We will develop, characterize and use in vivo, high efficiency delivery systems for ASO and ASG. Two delivery systems will be used: 1) previously described liposomes that deliver their contents into the cytoplasm of cells, 2) a novel approach which directly attaches targeting ligands to plasmid DNA. For the second approach we have synthesized bis-acridine intercalators that contain nuclear localization peptide sequences (NLS) or ligands for surface receptors. The affinity of the intercalator-ligands for DNA and the avidity of the ligand-modified DNA for the target receptor, as well as its extent of internalization, will be measured on target cells. Distribution of radiolabeled target plasmids will be measured following i.v. administration in mice. Since transfection frequency is limited by nuclear entry of DNA, NLS-acridine conjugates will be attached to DNA to increase nuclear uptake. Factors that control targeting of the modified DNA into the nucleus will be studied both in isolated nucleii and following microinjection of the complex into cells. The influence of increased plasmid nuclear localization on gene expression will be quantitated. We will study how macrophages present liposomal antigen in vivo to demonstrate the applicability of the approach for pharmacodynamic studies. Macrophages are absolutely required for the in vivo immune response to a liposomal antigen but it is not known if liposomal antigen is presented directly to T cells by macrophages or if an intervening cell is involved. As expression of the major histocompatibility complex II (MHC) on the macrophage is required for direct antigen presentation to T cells; suppression of macrophage MHC would differentiate between the alternatives. ASO and ASG will be constructed to inhibit the MHC expression in murine P388D1 macrophages (H-2d) and the effect of inhibition will be measured for in vitro presentation of ovalbumin. MHC surface expression and message levels will be quantitated. Conditions that inhibit MHC II expression in vitro will be used to study the response to liposomal ovalbumin in DBA/2 mice (syngenic with P388D1). The antisense approach will also be used to inhibit gene expression in liver, a major organ of drug metabolism. We will define the factors that influence antisense suppression of the alpha- 1-antitypsin gene (AP) in hepatocytes. The role of targeting ligand and carrier type on the extent and duration of inhibition will be followed by measuring serum levels of AP and correlated with AP message levels in the liver. Successful completion of this research should provide a valuable new tool for pharmacokinetic/dynamic studies. Moreover a high efficiency ASG delivery system would have obvious applications in gene therapy.
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