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Gene Expression Following Direct Injection of DNA into Liver

1994; Mary Ann Liebert, Inc.; Volume: 5; Issue: 12 Linguagem: Inglês

10.1089/hum.1994.5.12-1477

1557-7422

M. Anne Hickman, Robert W. Malone, Karin Lehmann‐Bruinsma, Tracey R. Sih, Daren L. Knoell, Francis C. Szoka, Rosemary L. Walzem, Don M. Carlson, Jerry S. Powell,

Virus-based gene therapy research

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), β-galactosidase (β-Gal) activity was evaluated in situ using plasmid DNA encoding Lac Z (pCMVβ), and serum concentration of secreted human α-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. β-Gal staining demonstrated that the majority of transfected hepatocytes were located near the injection site. Significant concentrations of human α-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. Numerous approaches exist for gene transfer into liver. This study describes a technique for the direct intrahepatic injection of DNA that leads to significant gene expression. The efficiency of gene delivery and expression was characterized in rats and cats by quantitative luciferase activity (pCMVL), in situ β-galactosidase (β-Gal) staining (pCMVβ), and measurement of serum concentrations of secreted human α-1-antitrypsin (pRC/CMV-sHAT). Direct injection of pCMVL resulted in maximal luciferase expression at 24–48 hr. (β-Gal staining demonstrated that the majority of transfected hepatocytes were located near the injection site. Significant concentrations of human α-1-antitrypsin were detected in the serum of all animals injected with pRC/CMV-sHAT. This study shows that direct DNA injection is a reliable and simple alternative for hepatic gene transduction.