Emulsiones

ARTICLE

doi:10.1016/j.ymthe.2005.06.477

Lipid Emulsions Potently Increase Transgene Expression in Hepatocytes after Adenoviral Transfer
Jan Snoeys,1 Geertrui Mertens,1 Joke Lievens,1 Theo van Berkel,2 Desire Collen,1 ´ ´ 2 1,* Erik A. L. Biessen, and Bart De Geest
2 1 Center for Molecular and Vascular Biology, University of Leuven, Campus Gasthuisberg, Herestraat 49, 3000 Leuven, BelgiumDivision of Biopharmaceutics, Leiden/Amsterdam Center for Drug Research, Leiden University, Gorlaeus Laboratories, Leiden, The Netherlands

*To whom correspondence and reprint requests should be addressed. Fax: +32 16 345990. E-mail: Bart.Degeest@med.kuleuven.be.

Available online 19 August 2005

Elimination of Kupffer cells by cytotoxic clodronate liposomes increases transgene expression inthe liver after adenoviral transfer. Here, we demonstrate that empty L-A-phosphatidylcholine liposomes block uptake of vectors in the reticuloendothelial cells of the liver and increase human apolipoprotein (apo) A-I (approved gene symbol apo A-I) expression in C57BL/6 (1.3-fold) and Balb/c mice (3.1-fold) to the same extent as clodronate liposomes (1.5- and 3.4-fold, respectively). A similarelevation of human apo A-I levels was induced by the lipid emulsion Intralipid (1.5- and 2.8fold in C57BL/6 and Balb/c mice, respectively). Not only Kupffer cells but also hepatic sinusoidal endothelial cells (HSEC) constitute the reticuloendothelial cells of the liver. The uptake of adenoviral vectors 1 h after transfer in C57BL/6 mice was 2.9-fold lower in Kupffer cells than in HSEC. In contrast,Kupffer cell uptake in Balb/c mice was 2.6-fold higher than in HSEC. Vector uptake in reticuloendothelial cells of the liver was reduced and transgene expression was increased in splenectomized and Rag2-deficient Balb/c mice but not in splenectomized and Rag1-deficient C57BL/6 mice. In conclusion, lipid emulsions for parenteral clinical use block uptake of adenoviral vectors by thereticuloendothelial cells of the liver and potently increase transgene expression. Key Words: gene therapy, adenovirus, liver, Kupffer cells, splenectomy, clodronate, lipid emulsions, intravenous, Rag2 protein, mouse, hepatocyte, endothelial cells

INTRODUCTION
Intravenous administration of adenoviral vectors leads to preferential transduction of the liver in mice, rats, and monkeys. Parenchymal cells comprise66% of liver cells, whereas sinusoidal cells constitute 33% of liver cells [1– 3]. The efficiency of adenoviral transfer to parenchymal liver cells is hampered by vigorous uptake of viral particles by tissue macrophages. Depletion of Kupffer cells in the liver and macrophages in the spleen by intravenous administration of clodronate liposomes results in increased transgene expression inparenchymal liver cells [4–6]. However, in addition to Kupffer cells, hepatic sinusoidal endothelial cells (HSEC)1 constitute a second major component of the hepatic reticuloendo-

Abbreviations: AdA-I, E1,E3,E4-deleted adenoviral vector containing a 1.5-kb human a1-antitrypsin promoter upstream of the genomic human apolipoprotein A-I sequence and four copies of the human apolipoprotein E enhancer, apo,apolipoprotein, HSEC, hepatic sinusoidal endothelial cells, NPC, nonparenchymal liver cells, PC, parenchymal liver cells.

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thelial system. Kupffer cells and HSEC constitute approximately 20 and 70%, respectively; stellate cells 10%; and pit cells less than 1% of the sinusoidal cells [3,7,8]. The contribution of HSEC to adenoviral vector scavenging after intravenous injection has not beeninvestigated until now. These microvascular endothelial cells have a unique phenotype characterized by the absence of a basal lamina, the presence of fenestrae, a considerable endocytic capacity, and a unique function as antigen-presenting cells of soluble antigens to CD4+ T cells and to CD8+ T cells by cross presentation [9]. HSEC can endocytose particles up to 230 nm [10] and thus potentially...