Delivery systems for gene therapy

Abstract

Introduction of foreign genes into mammalian cells in vitro has been accomplished previously by a variety of methods. The few techniques that have been developed for transfection of mammalian cells in vivo, are technically difficult or lack cell specificity. We have developed a soluble, targetable DNA carrier system consisting of an asialoglycoprotein covalently coupled to a polycation. The strategy was based on: 1) the presence of unique receptors on hepatocytes which internalize galactose-terminal (asialo-)glycoproteins; 2) polycations can bind DNA in a non-covalent, non-damaging interaction. Using chloramphenicol acetyltransferase (CAT) as a marker gene, specific delivery and expression of CAT was demonstrated in vitro using asialoglycoprotein receptor (+) and (-) cell lines. Intravenous injection of conjugate-DNA complexes in rats resulted in detection of CAT DNA sequences in liver 10 min later by dot blots with a CAT cDNA probe. CAT enzyme activity 24 hrs later was found specifically in liver but no other tissues or control livers. Targeted hepatic CAT expression was transient, maximal at 24 hrs but declined to barely detectable levels by 96 hrs. Persistent foreign gene expression was achieved by injection of DNA complex followed by 67% partial hepatectomy. High levels of hepatic CAT activity were detected through 11 weeks post-hepatectomy. The data indicate that a targetable gene delivery system can permit in vivo expression of an exogenous gene after simple intravenous injection. The foreign gene expression can be enhanced and made to persist by induction of hepatocyte replication.