Naked plasmid DNA transfer to the porcine liver using rapid injection with large volume

Abstract

The naked plasmid DNA transfer method of rapid injection with large volume has been useful for gene therapy in experimental study. However, only small animals like rodents have usually been reported on. In this study, the authors attempted to transfect naked plasmid DNA to the porcine liver by modified hydrodynamic method. We decided to transfer plasmid DNA to a part of the liver using the angio-catheter to reduce the liver damage. To discern the condition of injection, naked plasmid DNA-encoding green fluorescent protein (GFP) was transferred for use as a marker gene. The GFP gene expression was markedly observed in gene-transferred pig livers. In large animals, not only the naked gene quantity, the solution volume containing the plasmid DNA and the injection speed, but also the additional treatments of the portal vein and the hepatic artery preparation were crucial. We found that the following injection condition were needed: plasmid DNA, 3 mg; the solution volume, 150 ml and the injection speed, 5 ml/s. The portal vein and the hepatic artery were clamped during gene delivery and the blood flow of the portal vein was flushed out using normal saline. Cytotoxic T-lymphocyte antigen 4-immunoglobulin (CTLA4-Ig) gene was used to test for secretory protein. CTLA4-Ig gene was injected with a large volume of solution via the hepatic vein to the left outer lobe of the liver selectively. CTLA4-Ig was detected in the pig blood at a maximum serum level of 161.7 ng/ml 1 day after gene transfer, and the CTLA4-Ig was detected for several weeks. Our new technique of inserting a catheter into only a selected portion of the liver reduced liver toxicity and increased gene transfer efficiency. This is the first report of successful gene transfer, using a hydrodynamic method, to the segmental liver in pigs, and achieved more than enough secretory protein for the clinically therapeutic level in pigs.