Nonenzymatic glycosylation of poly-L-lysine: a new tool for targeted gene delivery.
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 cross-linked 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 epsilon-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-beta 1 complementary DNA.(ABSTRACT TRUNCATED AT 250 WORDS)
Martinez-Fong
D
D
Research and Development Service, Veterans Affairs Medical Center, Memphis, Tennessee.
Mullersman
J E
JE
Purchio
A F
AF
Armendariz-Borunda
J
J
Martinez-Hernandez
A
A
D000818:Animals; D001212:Asialoglycoproteins / Q000378:metabolism; D015152:Blotting, Northern; D006528:Carcinoma, Hepatocellular / Q000378:metabolism; D005260:Female; D018014:Gene Transfer Techniques; D005822:Genetic Vectors; D006031:Glycosylation; D007785:Lactose / Q000378:metabolism; D008099:Liver / Q000378:metabolism; D008113:Liver Neoplasms / Q000378:metabolism; D010957:Plasmids; D011107:Polylysine / Q000378:metabolism*; D012333:RNA, Messenger / Q000378:metabolism; D051381:Rats; D017207:Rats, Sprague-Dawley; D014162:Transfection; D016212:Transforming Growth Factor beta / Q000235:genetics
D001212:Asialoglycoproteins; D012333:RNA, Messenger; D016212:Transforming Growth Factor beta; D011107:Polylysine; D007785:Lactose