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High efficiency gene transfer to liver with naked DNA.
DJ Vargo, G Zhang, V Budker, JA Wolff, SJ Knechtle.
Departments of Surgery and Pediatrics, University of Wisconsin, Madison, WI.

Efficient gene transfer to hepatocytes without use of viral vectors would have great advantage for liver targeted gene therapy. We have utilized isolated liver perfusion in vivo to transfect hepatocytes with naked plasmid DNA. This technique involves total vascular isolation of the liver, with delivery of the plasmid via either the portal vein (PV), IVC, or bile duct, with or without outflow occlusion. The plasmids pCILuc (luciferase gene) or pCILacZ (ß-galactosidase gene) were injected in normal saline with 15% mannitol. Animals were sacrificed at 24 hours, livers were harvested and prepared for analysis by homogenation and extraction of the enzyme, or by tissue staining for X-Gal. Results in the mouse (expressed as ug luciferase/liver) with 100 ug plasmid showed increased expression if there was outflow occlusion from the liver. Results in a rat model with 750 ug plasmid were similar.

PV+occl.       PV-occl.      IVC+occl.        IVC-occl.
Mouse           3.73           0.005          17.34            2.83
Rat             53.5                           1.50

Bile duct injection in the mouse and rat yielded 15.39 ug/liver and 1.3 ug/liver, respectively. Of variables measured (DNA concentration, duration of injection, volume of infusate), the amount of intraparenchymal pressure generated during injection of the plasmid seemed the most important, with the optimal pressure being 31-40mm Hg. The effect of pressure plateaued above 40mm Hg.

intraparenchymal pressure (mm Hg)        mean light units (X 10^{9})
             10-20                                    51
             21-30                                   121
             31-40                                  1910
             >40                                     580

ß-galactosidase activity measured with X-Gal staining showed uptake and activity distributed fairly evenly in the mouse liver, while rat liver showed a preference toward peri-acinar positivity with PV injection and central vein positivity with IVC injection. In summary, we have developed a direct DNA delivery technique which allows hepatocytes to be transfected in vivo, does notrequire removal of any liver tissue to be effective, and provides levels of gene expression that are 10-100 times higher than previously reported with other direct gene transfer techniques.