NONENZYMATIC GLYCOSYLATION OF POLY-L-LYSINE - A NEW TOOL FOR TARGETEDGENE DELIVERY

The basic approach in targeted gene delivery relies on the formation o f a complex between a vector and a molecule that will be selectively i nternalized by the target cells. In the case of hepatocytes, asialogly coproteins are convenient targeting molecules because of the high affi nity and avidity of the hepatocyte galactose receptor. In this system, poly-L-lysine is cross-linked to an asialoglycoprotein, and the resul ting conjugate is complexed with the expression vector (DNA). The elec trostatic binding between DNA and poly-L-lysine-asialoglycoprotein ens ures delivery of the intravenously injected complex to the liver, wher e it is subjected to endocytosis by hepatocytes. However, the poly-L-l ysine-asialoglycoprotein complexes tend to be unstable, of limited sol ubility and of searched carbohydrate content. For these reasons we sea rched for a simpler alternative. We exploited the known capacity of re ducing sugars to be reductively coupled to the epsilon-amino groups in proteins and used lactose to obtain poly-L-lysine with ''exposed'' ga lactose. Glycosylation with sodium cyanoborohydride at high pH in bora te buffer is a simple, reproducible procedure. The ''lactosylated'' po ly-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 comple xes by transfecting hepatoma cells (HepG2) in culture. For these exper iments we used a pRc/cytomegalovirus eukaryotic expression vector cont aining a mutant TGF-beta 1 complementary DNA. On Northern-blot analysi s, cells transfected with lactosylated poly-L-lysine expressed 10 to 2 0 times more TGF-beta 1 messenger RNA than did cells transfected with the same plasmid coupled to asialofetuin-poly-L-lysine. Therefore glyc osylated poly-L-lysine is a simple, highly effective alternative to po ly-L-lysine-asialoprotein complexes. Furthermore the use of different disaccharides may permit targeting of the complexes to different cell types.