Nonenzymatic glycosylation of poly-l-lysine: A new tool for targeted gene delivery

Authors

  • Daniel Martinez-Fong,

    1. Pathology and Laboratory Medicine Service, Advanced Tissue Sciences, La Jolla, California 92037
    2. Departments of Pathology, Advanced Tissue Sciences, La Jolla, California 92037
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  • Jerald E. Mullersman,

    1. Pathology and Laboratory Medicine Service, Advanced Tissue Sciences, La Jolla, California 92037
    2. Departments of Pathology, Advanced Tissue Sciences, La Jolla, California 92037
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  • Anthony F. Purchio,

    1. University of Tennessee Medical College, Memphis, Tennessee 38104, Advanced Tissue Sciences, La Jolla, California 92037
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  • Juan Armendariz-Borunda,

    1. Research and Development Service, Advanced Tissue Sciences, La Jolla, California 92037
    2. Department of Medicine, Veterans Affairs Medical Center, Advanced Tissue Sciences, La Jolla, California 92037
    3. Departments of Medicine, Advanced Tissue Sciences, La Jolla, California 92037
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  • Antonio Martinez-Hernandez M.D.

    Corresponding author
    1. Pathology and Laboratory Medicine Service, Advanced Tissue Sciences, La Jolla, California 92037
    2. Departments of Pathology, Advanced Tissue Sciences, La Jolla, California 92037
    • Pathology and Laboratory Medicine Service, Veterans Affairs Medical Center, 1030 Jefferson Avenue, Memphis, TN 38104
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Abstract

The basic approach in targeted gene delivery relies on the formation of a complex between a vector and a molecule that will be selectively internalized by the target cells. In the case of hepatocytes, asialoglycoproteins are convenient targeting molecules because of the high affinity and avidity of the hepatocyte galactose receptor. In this system, poly-l-lysine is crosslinked to an asialoglycoprotein, and the resulting conjugate is complexed with the expression vector (DNA). The electrostatic binding between DNA and poly-l-lysine—asialoglycoprotein ensures delivery of the intravenously injected complex to the liver, where it is subjected to endocytosis by hepatocytes. However, the poly-l-lysine—asialoglycoprotein complexes tend to be unstable, of limited solubility and of fixed carbohydrate content. For these reasons we searched for a simpler alternative. We exploited the known capacity of reducing sugars to be reductively coupled to the -amino groups in proteins and used lactose to obtain poly-l-lysine with „exposed” galactose. Glycosylation with sodium cyanoborohydride at high pH in borate buffer is a simple, reproducible procedure. The „lactosylated” poly-l-lysine has proved very stable, highly soluble and easily bound to plasmids. In a set of experiments we compared the asialofetuin—poly-l-lysine vector complexes with lactosylated poly-l-lysine vector complexes by transfecting hepatoma cells (HepG2) in culture. For these experiments we used a pRc/cytomegalovirus eukaryotic expression vector containing a mutant TGF-β1 complementary DNA. On Northern-blot analysis, cells transfected with lactosylated poly-l-lysine expressed 10 to 20 times more TGF-β1 messenger RNA than did cells transfected with the same plasmid coupled to asialofetuin—poly-l-lysine. Therefore glycosylated poly-l-lysine is a simple, highly effective alternative to poly-l-lysine—asialoprotein complexes. Furthermore the use of different disaccharides may permit targeting of the complexes to different cell types. (Hepatology 1994;20:1602–1608).

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