Kenji Kawabata

The Fate of Plasmid DNA After Intravenous Injection in Mice: Involvement of Scavenger Receptors in Its Hepatic Uptake

AbstractPurpose. We examined the stability and disposition characteristics of a naked plasmid DNA pCAT as a model gene after intravenous injection in mice to construct the strategy of in vivo gene delivery systems. Methods. After the injection of pCAT to the mice, stability, tissue distribution, hepatic cellular localization, and effect of some polyanions on the hepatic uptake were studied. Results. The in vitro study demonstrated that the pCAT was rapidly degraded in mouse whole blood with a half-life of approximately 10 min at a concentration of 100 µg/ml. After intravenous injection, pCAT was degraded at a significantly faster rate than that observed in the whole blood, suggesting that pCAT in vivo was also degraded in other compartments. Following intravenous injection of [32P] pCAT, radioactivity was rapidly eliminated from the plasma due to extensive uptake by the liver. Hepatic accumulation occurred preferentially in the non-parenchymal cells. The hepatic uptake of radioactivity derived from [32P] pCAT was inhibited by preceding administration of polyanions such as polyinosinic acid, dextran sulfate, maleylated and succinylated bovine serum albumin but not by polycytidylic acid. These findings indicate that pCAT is taken up by the liver via scavenger receptors on the non-parenchymal cells. Pharmacokinetic analysis revealed that the apparent hepatic uptake clearance was fairly close to the liver plasma flow. Conclusions. These findings provide useful information for the development of delivery systems for in vivo gene therapy.

In Vivo Disposition Characteristics of Plasmid DNA Complexed with Cationic Liposomes

To control disposition and hence gene expression, we investigated the disposition characteristics of plasmid DNA complexed with the cationic liposomes Lipofectin and LipofectACE after intravenous injection in mice via the tail vein. The optimum ratios of DNA and liposome complexes were selected through in vitro cytotoxicity and transfection studies. The highest transfection was found at the DNA:liposome ratio of 1:5 w/w. Hence, this ratio was used for in vivo disposition studies, and the distribution patterns were compared with that of naked pCAT. Following intravenous injection of [32P] pCAT, radioactivity was rapidly eliminated from plasma and approximately 60% of the dose was taken up by the liver within 1.5 min. In the case of LipofectACE samples, radioactivity elimination from plasma was equally rapid, but its accumulation was observed in both the liver (35%) and the lung (45%). For Lipofectin samples, radioactivity was initially accumulated in both the liver (55%) and the lung (25%), but lung accumulation was not sustained beyond 5 min after administration. Both liposomal samples showed in vivo gene expression in the lung, heart, kidney and spleen, but not in the liver. Thus, the present study demonstrated that disposition and gene expression of pCAT can be controlled by complex formation with liposomes.

Disposition Characteristics of Plasmid DNA in the Single-pass Rat Liver Perfusion System

AbstractPurpose. To define the hepatic uptake mechanism of a plasmid DNA, we quantitated the uptake of pCAT (plasmid DNA encoding chloramphenicol acetyltransferase reporter gene fused to simian virus 40 promoter), a model plasmid, after a single pass through the perfused rat liver using albumin- and erythrocyte-free Krebs-Ringer bicarbonate buffer (pH 7.4). Methods. [32P]pCAT was introduced momentarily into this system from the portal vein as a bolus input or constant infusion mode, and the outflow patterns and hepatic uptake were evaluated using statistical moment analysis. Results. The venous outflow samples had electrophoretic bands similar to that of the standard pCAT, suggesting that the plasmid is fairly stable in the perfusate during liver perfusion. In bolus experiments, pCAT was largely taken up by the liver and the uptake was decreased with increase in injected dose. Statistical moment analysis against outflow patterns demonstrated that the apparent volume of distribution of pCAT was greater than that of human serum albumin, indicating a significant reversible interaction with the tissues. The results of collagenase perfusion experiments suggest that the hepatic accumulation of pCAT occurred preferentially in the nonparenchymal cells (NPC). The amount of total recovery in the liver decreased substantially by preceding administration of polyinosinic acid, dextran sulfate, succinylated bovine serum albumin, but not by polycytidylic acid. This suggests that pC AT is taken up by the liver via scavenger receptors for polyanions on the NPC. In constant infusion experiments, the presence of 2,4-dinitrophenol and NH4C1 caused a significant increase in the outflow concentration of [32P]pCAT and decrease by half in the total hepatic recovery than that of plasmid DNA administered alone, suggesting that plasmid DNA may undergo internalization by the NPC. Conclusions. The liver plays an important role in the elimination of plasmid DNA and a successful delivery system will be required to avoid its recognition by the scavenger receptors on the liver NPC.

Pharmacokinetics and targeted delivery of proteins and genes

The effectiveness of various approaches for controlling in vivo disposition of proteins and genes was compared based on pharmacokinetic analysis. The potential of introduction of galactose or mannose residues aiming at receptor-mediated endocytosis, succinylation to be recognized by a scavenger receptor, and cationization for universal electrostatic interaction were characterized using model proteins. Corresponding to the results, a superior therapeutic effect was shown with derivatives of superoxide dismutase against hepatic and renal ischemia/reperfusion injury. A similar approach was adopted for plasmid DNA and oligonucleotide and their rapid degradation in the blood pool and preferential uptake by the liver after intravenous injection were characterized by pharmacokinetic analysis. The effects of incorporation into cationic liposomes and conjugation with macromolecules on their in vivo distribution were also elucidated.

Non-polarized secretion of mouse interferon-β from gene-transferred human intestinal Caco-2 cells

AbstractPurpose. The intestinal epithelium is considered to be a feasible target for somatic gene therapy. To this end, Caco-2 cells derived from human colon carcinoma were transfected with a mouse interferon-β (IFN-β) expression vector and several stable sublines were established; this hetero-specific cytokine allows unexpected cellular effects to be avoided. Using the highest mouse IFN-β-producing sublines, the mode of IFN secretion was examined. Methods. The secretion polarity of mouse IFN-β in its gene-transduced Caco-2 sublines was studied in a bicameral culture system in which the chambers were separated by microporous filters. Results. Mouse IFN-β was secreted to the same extent from both apical and basolateral surfaces of the transduced cells regardless of cell aging. Conclusions. These results suggest that in the intestinal epithelium exogenous gene products such as IFNs can be delivered to both the luminal and blood sides in vivo. Thus, the intestinal epithelium may be suitable for systemic and local delivery of therapeutic proteins by gene transfer.

A physiological role of interferon (IFN)-β derived from tumor: tumor growth of a mouse bladder carcinoma line MBT-2 is partially suppressed by autocrine IFN-β

Although some tumor cells endogenously produce a wide variety of cytokines, their physiological roles remain to be fully understood. In this study, we found that mouse subcutaneous tumor induced by inoculation of bladder tumor MBT-2 cells into syngeneic mice secreted a significant amount of interferon (IFN), whereas the cells exhibited no IFN production in in vitro cell culture. Typing experiment using IFN-specific neutralizing antibodies showed that the tumor-derived IFN was exclusively β type. Since the MBT-2 tumor tissues were homogenous and not infiltrated by immune cells, MBT-2 cells themselves were considered to be IFN-β producers. By intraperitoneal injection of neutralizing anti-IFN-β antibodies into MBT-2 cell-inoculated mice, the tumor growth was substantially precipitated and survival days of the tumor-bearing mice were shortened. As the in vitro cell growth of MBT-2 cells was dose-dependently inhibited by IFN-β, it was suggested that apparent immunogenicity of MBT-2 tumor is partially mediated by tumor suppression by autocrine IFN-β.