Galactosylated Histone-Mediated Gene Transfer and Expression

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Abstract
We have developed a novel, highly efficient DNA delivery system to accomplish gene transfer through the asialoglycoprotein receptor-mediated endocytosis pathway. Natural nuclear DNA-binding proteins, the histones (H1, H2a, H2b, H3, and H4), were modified and used as receptor-targeted DNA carriers. Galactosylated with a coupling agent, 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide, the histones and albumin were conjugated to DNA and then used to transfect HepG2 cells, which display the asialoglycoprotein receptor. The extent of galactosylation was determined for all histone subgroups and albumin with 14C-labeled galactose. A reporter gene for the bacterial chloramphenicol acetyltransferase (CAT), under the control of the 5′ long terminal repeat (LTR) of Rous sarcoma virus, was used for comparisons of transfection efficiency of various carrier proteins. The CAT activity resulting from histone H1-mediated transfection was 1.66 unit per 106 cells, the highest among histone subgroups. The galactyosylated histone H1 was also eleven times more effective than the asialo-orosomucoid-polylysine. Ten galactosyl units are attached to histone H1 by the galactosylation reaction. Differences in the extent of galactosylation could not explain different transfection efficiencies among various proteins studied in this report. Treatment with galactose oxidase abolished the transfection ability of both the galactosylated histone H1 and asialo-orosomucoid. The intrinsic DNA-binding domains and nuclear location signal sequences are unique to histones as receptor-targeted DNA carriers, and are advantageous for effective gene delivery. High-level expression of reporter gene has been achieved in hepatoma cells with galactosylated histone-mediated transfection. Compared with other receptor-targeted gene delivery systems (Wu et al., 1989; Wagner et al., 1990), the modified, multifunctional histones offer several unique features, including their intrinsic DNA-binding domains and nuclear location signals. This paper reports a simple galactosylation reaction for proteins and the use of the modified proteins as vehicles for DNA delivery targeting the asialoglycoprotein receptor. When used to deliver an expression vector for the chloramphenicol acetyltransferase to the hepatoma HepG2 cells, the transfection efficiency of the galactosylated histone H1 was superior to other modified histone subgroups and nonnuclear proteins. The natural ability of histones to compact DNA and to enter the cell nucleus may be responsible for the high efficiency of histone H1-mediated gene transfer. However, it is not clear why other histone subgroups were less effective than the histone H1.