Evaluation of gene promoters for liver expression by hydrodynamic gene transfer

Objective. Gene therapy represents a promising treatment for hepatic disease. Most approaches today use viral methods to target tissues. While nonviral 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-1 alpha (EF1 alpha), and phosphoglycerokinase (PGK) promoters were subcloned into plasmids with a luciferase reporter gene. In vitro calcium phosphate-mediated transfection of 2 X 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 (FVIII)-deficient mice were similarly injected with CBA-or EF1 alpha-promoted constructs containing the FVIII heavy and light chains and expression was examined. Results. In vitro transfection demonstrated a hierarchy of expression: hCMV-intron > CAG > EF1 alpha > 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 alpha, hCMV, and PGK constructs respectively. FVIII plasmid injected hemophilic mice demonstrated higher levels of FVIII expression with CAG versus EF1 alpha, confirming the reporter gene studies. All FVIII-deficient mice injected with EF1 alpha-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. (C) 2008 Elsevier Inc. All rights reserved.