Neonatal gene therapy has the potential to ameliorate abnormalities before disease onset. Our gene knockout of arginase I (AI) deficiency is characterized by increasing hyperammonemia, neurological deterioration, and early death. We constructed a helper-dependent adenoviral vector (HDV) carrying AI and examined for correction of this defect. Neonates were administered 5 x 10(9) viral particles/g and analyzed for survival, arginase activity, and ammonia and amino acids levels. The life expectancy of arg(-/-) mice increased to 27 days while controls died at 14 days with hyperammonemia and in extremis. Death correlated with a decrease in viral DNA/RNA per cell as liver mass increased. Arginase assays demonstrated that vector-injected hepatocytes had ~20% activity of heterozygotes at 2 weeks of age. Hepatic arginine and ornithine in treated mice were similar to those of saline-injected heterozygotes at 2 weeks, whereas ammonia was normal. By 26 days, arginase activity in the treated arg(-/-) livers declined to <10%, and arginine and ornithine increased. Ammonia levels began increasing by day 25, suggesting the cause of death to be similar to that of uninjected arg(-/-) mice, albeit at a later time. These studies demonstrate that the AI deficient newborn mouse can be temporarily corrected and rescued using a HDV.

Gene therapy represents a promising treatment for hepatic disease. Most approaches today utilize viral methods to target tissues. While non-viral gene therapy is less prominent, hydrodynamic gene delivery represents a promising approach to direct gene expression to the liver. The purpose of the present study was to evaluate promoters for efficient gene expression in hepatocytes in vivo by hydrodynamic delivery and to test the findings in a model of hemophilia A. Materials and Methods-Human cytomegalovirus (hCMV), chicken beta-actin/CMV enhancer (CAG), elongation factor-1alpha (EF1α), and phosphoglycerokinase (PGK) promoters were subcloned into plasmids with a luciferase reporter gene. In vitro calcium phosphate-mediated transfection of 2×10 5 HEK 293 cells was followed by in vivo whole animal bioluminescence and luminometry after hydrodynamic tail vein injection of plasmid DNA. Six-month-old FVB factor VIII-deficient mice were similarly injected with CBA-or EF1α-promoted constructs containing the factor VIII heavy and light chains and expression was examined. Results-In vitro transfection demonstrated a hierarchy of expression: hCMV-intron> CAG>EF1α>hCMV>>PGK. In vivo luminometry demonstrated that the CAG construct produced 2.6x, 3.0x, 3.4x, and >1000x the expression of the hCMV-intron, EF1 α, hCMV, and PGK constructs respectively. Factor VIII (FVIII) plasmid injected hemophilic mice demonstrated higher levels of factor VIII expression with CAG versus EF1α, confirming the reporter gene studies. All FVIIIdeficient mice injected with EF1 α-FVIII or CAG-FVIII plasmids survived after tail clipping.Conclusions-The CAG promoter/enhancer combination is an excellent alternative to the human CMV promoter for hydrodynamic gene delivery to the liver.

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