358. Non-Viral/Minicircle Vector Transduction of Hepatocytes in Small Pigs By Hydrodynamic Intraportal Injection

Abstract

Non-viral and non-plasmid-based vectors that express a therapeutic protein from an episomal element have the potential of robust and sustained transgene expression and at the same time bear a very low risk of insertional mutagenesis. While hydrodynamic injection into the tail vein of mice to target the liver is an efficient but experimental approach, delivery of naked DNA/minicircle vectors into large animals and humans remains a challenge. Here we show successful hepatocyte transfection in domestic small pigs treated immediately after weaning with portal vein catheterization and hydrodynamic injection of naked DNA/minicircle vectors expressing the luciferase gene from a liver-specific promoter. We established a surgical method allowing hydrodynamic portal vein pressurization up to 90 mmHg and infusion of naked DNA in pigs with long-term survival. No acute adverse effects such as changes in liver transaminases or signs of liver cell damage were observed. Stable hepatocyte transfections at 10 and 28 days in single experiments were found in up to 60% of samples (45/75 were PCR-positive for minicircle-DNA) and 13% of the positive specimens (6/45) showed low but stable luciferase expression with up to two vector copies per diploid hepatocyte genome. While further optimization to enhance transfection efficiency and transgene expression is required, we conclude that hydrodynamic gene delivery using minicircle vectors in small pigs is a safe procedure for stable transfection of liver cells as a prerequisite to potentially treat infants with genetic liver diseases.