We have reported that a rapid tail vein injection of a large volume of plasmid DNA solution into a mouse results in high level of transgene expression in the liver. Gene transfer efficiency of this hydrodynamics-based procedure is determined by the combined effect of a large volume and high injection speed. Here, we show that the hydrodynamic injection induces a transient irregularity of heart function, a sharp increase in venous pressure, an enlargement of liver fenestrae, and enhancement of membrane permeability of the hepatocytes. At the cellular level, our results suggest that hepatic delivery by the hydrodynamic injection is accomplished by the generation of membrane pores in the hepatocytes.

The liver is an important target organ for gene transfer due to its large capacity for synthesizing serum proteins and its involvement in numerous genetic and acquired diseases. Previously, we and others have shown that an efficient gene transfer to liver cells in vivo can be achieved by an intravenous injection of plasmid DNA using a hydrodynamics-based procedure. In this study, we systematically characterized the expression of transgene encoding a secretory protein in mouse. Using human ␣1-antitrypsin (hAAT) gene as a reporter, we demonstrate that the serum level of hAAT can reach as high as 0.5 mg/ml by a simple

Eleven structural analogues of two known cationic lipids, N-[1-(2, 3-dioleyloxy)propyl]-N,N,N-trimethylammonium chloride (DOTMA) and N-[1-(2,3-dioleoyloxy)propyl]-N,N,N-trimethylammonium chloride (DOTAP) were synthesized and utilized to evaluate the structural characteristics of DOTMA for its high intravenous transfection activity. Using a CMV-driven expression system and luciferase gene as a reporter, the transfection activity of these analogues was evaluated in mice using tail vein injection. Results concerning the structure-activity relationship with regard to the influence of the backbone, relative position between head group and the hydrophobic chains on the backbone, linkage bonds, as well as the composition of the aliphatic chains revealed that cationic lipids which give a higher in vivo transfection activity share the following structural characteristics: (1) cationic head group and its neighboring aliphatic chain being in a 1,2-relationship on the backbone; (2) ether bond for bridging the aliphatic chains to the backbone; and (3) paired oleyl chains as the hydrophobic anchor. Cationic lipids without these structural features had lower in vivo transfection activity. These structural characteristics, however, did not significantly influence their in vitro transfection activity. The contribution that cationic lipids make to the overall in vivo transfection activity is likely to be determined by the structure of DNA/lipid complexes and by the outcome of the interaction between the DNA/lipid complexes and blood components upon intravenous administration.