Time course of gene expression after plasmid DNA gene transfer to the liver

Background High levels of expression in hepatocytes can be achieved after i ntraportal delivery of plasmid DNA vectors with up to 10% of all liver cell s transfected. CMV promoter-driven expression is very high on Day 1 after i njection, but is diminished strongly by Day 2. Expression slowly declines a fter 1 week. We describe experiments aimed at elucidating the reasons for t his rapid decline in transgene expression. Methods Histological methods were used to determine the presence and extent of Liver damage and hepatocyte proliferation. Viral and liver-specific pro moters were tested to study promoter shut-off, Southern blotting was perfor med to determine the loss of the pDNA vector over time, and several mouse m odels were used to study the host immunological response. Results pDNA is lost rapidly early after injection, but remains at a relati vely stable copy number after Day 4. Southern blotting experiments showed t hat plasmid DNA could be detected for at least 12 weeks after injection (0. 2 copies per genome). The early rapid decline of expression is promoter dep endent. A liver-specific albumin promoter resulted in similar levels of exp ression on Days 1 and 7, suggesting that promoter inactivation may be respo nsible for the instability of CMV promoter-driven expression. The slow decl ine in expression levels after 1 week appears to be the result of an immune response directed against the expressed transgene. Expression was much pro longed in immunosuppressed, immunodeficient, or antigen-tolerized mice. Conclusion The present data suggest that if promoter inactivation can be ov ercame, intravascular delivery of plasmid DNA could be a highly efficient, simple and non-toxic liver gene therapy approach. Intravascular delivery of pDNA allows for the rapid screening of novel expression vectors in vivo. C opyright (C) 2001 John Wiley & Sons, Ltd.