Gene Expression Following Direct Injection of DNA into Liver

Abstract

The liver is an attractive target tissue for gene therapy. Current approaches for hepatic gene delivery include retroviral and adenoviral vectors, liposome/DNA, and peptide/DNA complexes. This study describes a technique for direct injection of DNA into liver that led to significant gene expression. Gene expression was characterized in both rats and cats following injection of plasmid DNA encoding several different proteins. Luciferase activity was measured after injection of plasmid DNA encoding the luciferase gene (pCMVL), beta-galactosidase (beta-Gal) activity was evaluated in situ using plasmid DNA encoding Lac Z (pCMV beta), and serum concentration of secreted human alpha-1-antitrypsin was measured following injection of plasmid DNA encoding this protein (pRC/CMV-sHAT). Several variables, including injection technique, DNA dose, and DNA diluent, were investigated. Direct injection of pCMVL resulted in maximal luciferase expression at 24-48 hr. beta-Gal staining demonstrated that the majority of transfected hepatocytes were located near the injection site. Significant concentrations of human alpha-1-antitrypsin were detected in the serum of animals injected with pRC/CMV-sHAT. These findings demonstrate the general principle that direct injection of plasmid DNA into liver can lead to significant gene expression.

Hydrodynamics-based transfection in animals by systemic administration of plasmid DNA

“…Following this initial discovery, many studies have reported success in expressing exogenous genes using local regional administration. [18][19][20][21][22][23] In this report, we have demonstrated that an efficient gene transfer and high level of transgene expression can also be achieved by systemic administration of plasmid DNA in animals by a rapid injection of DNA in large volume (Figures 1 and 2). Up to 300 ng of transgene product per milligram of extracted protein was obtained from the liver of a mouse after a single injection of as little as 5 g of plasmid DNA (Figure 3).…”

“…[17][18][19][20][21][22][23] Compared with virus-mediated gene transfer systems, introduction of an exogenous gene into cells in the form of naked DNA has many obvious advantages. Preparation of DNA has become routine in many laboratories, and it is easy to obtain significant amounts of DNA without involving laborious procedures.…”

“…Since DNA is chemically and biologically stable, no sophisticated storage conditions are required. Since the initial report in 1990 of the successful expression of a reporter gene in muscle by the method of intramuscular injection of plasmid DNA, 17 there have been many in vivo studies demonstrating successful transgene expression in liver, [18][19][20] lung, 21 heart, 22 and skin 23 using a similar method. However, the major limitation of this method is that it requires a local regional administration or surgical procedures.…”

Long-term expression of human alpha1-antitrypsin gene in mouse liver achieved by intravenous administration of plasmid DNA using a hydrodynamics-based procedure

“…30 The livers were collected and immediately frozen on dry ice. Approximately 200 mg of liver sample were used for DNA extraction.…”

Highly efficient gene transfer into murine liver achieved by intravenous administration of naked Epstein–Barr virus (EBV)-based plasmid vectors

“…2 Naked pDNA transfection was also successful into other organs, such as heart, 3 lung, 4 skin, 5 and liver. 6 More recently, very high transgene expression was achieved in the liver by intravascular delivery of pDNA. Budker et al 7 and Zhang et al 8 administered pDNA into the liver via the portal vein, hepatic vein or the bile duct.…”