812. Hydroporation as the Mechanism Accounting for Efficient Gene Transfer by Hydrodynamic Delivery
Authors: Dexi Liu, Guisheng Zhang, Xiang Gao, Young K. Song, Regis Vollmer, Donna B. Stolz, Joshua Z. Gasiorowski, David A. Dean
Journal: Molecular Therapy (2004)

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We have reported that hydrodynamic delivery of plasmid DNA into a mouse results in a high level of transgene expression in many organs with the highest in the liver. Gene transfer efficiency by this procedure is dictated by a combined effect of large volume and high injection speed. The current study was designed to elucidate the underlying mechanism of the hydrodynamic delivery.

Using a catheter inserted to the point of vena cava through a renal vein in retrograde, we observed a sharp increase in venous pressure and then a rapid drop to a level of 10-15 mmHg above the original baseline pressure, followed by a much slower decline. Electrocardiogram (ECG) of a mouse undergoing the hydrodynamic injection showed an irregular cardiac rhythm, which lasted about 60 sec. The heart rate decreased from approximately 510 beats/min to 280 beats/min 3 sec after the injection and returned to normal in 2 min. SEM and TEM examination of the liver sections revealed partial disruption of endothelial lining of liver capillaries, enlargement of liver fenestrae and more importantly membrane pores on hepatocytes. Using Evans blue and reporter gene containing plsmids as a probe we found that these membrane pores were transient and resealed within a few min. We also demonstrated that proteins (beta-galactosidase), fluorescent dye (Evans blue), reporter plasmids and 125I-labeled DNA can all be hydroynamically delivered into hepatocytes with very similar efficiency. Using cytoplasmic microinjection technique and GFP as a reporter, we demonstrated that a large volume of DNA solution entering the cytoplasm helps transgene expression. For example, GFP positive cells increased from approximately 26% of injected cells (n=339) at 5-10% cytoplasmic volume to 50% at 50-70% of cytoplasmic volume (n=303).

We conclude from these results that hydrodynamic delivery is a physical process that does not involve membrane receptors for DNA transfer into cells. The mechanism underlying this efficient procedure can be described as follows. A rapid injection of a large bolus of DNA solution via the tail vein causes a transient cardiac congestion, resulting in an accumulation of the injected solution in the inferior vena cava. Subsequently, a high pressure develops in this venous region and causes a retrograde movement of the solution into the liver, an organ with enriched vascular structure and direct connection to the inferior vena cava. A large portion of DNA solution enters the liver via the hepatic vein under a hydrodynamic force, pushing the blood in hepatic circulation to move backward toward the portal blood vessels. As a consequence of this hydrodynamic influence (mechanic or/and pressure influence due to hydrodynamic injection) the pore sizes of the liver fenestrae are enlarged and the membrane pores on hepatocytes are generated. Consequently, DNA is driven into the hepatocytes and membrane of hepatocytes reseals with time, trapping the DNA molecules inside. In a short time, the cardiovascular system adapts to the volume load and normal circulatory homeostasis is restored.