Lentiviral vectors for efficient transduction of isolated primary quiescent hepatocytes

• Published in: Journal of hepatology Vol. 36; no. 4; pp. 459 - 465
• Main Authors: Seppen, Jurgen, Rijnberg, Martijn, Cooreman, Michel P, Oude Elferink, Ronald P.J
• Format: Journal Article
• Language: English
• Published: Oxford Elsevier B.V 01.04.2002; Elsevier
• Subjects: Anesthesia. Intensive care medicine. Transfusions. Cell therapy and gene therapy; Animals; Applied cell therapy and gene therapy; Biological and medical sciences; Carbamoyl-Phosphate Synthase (Ammonia) - metabolism; Cells, Cultured; Gene therapy; Gene Transfer Techniques; Genetic Vectors; Hepatitis B virus; Hepatitis B virus - genetics; Hepatocytes - enzymology; Hepatocytes - physiology; HIV - genetics; Human immunodeficiency virus; Lentivirus - genetics; Liver; Medical sciences; Mice; Microscopy, Fluorescence; Polypurine tract; Posttranscriptional regulatory element; Purine Nucleotides - genetics; Rats; RNA - metabolism; Trans-Activators - physiology; Transfusions. Complications. Transfusion reactions. Cell and gene therapy; Virion - genetics; Posttranscriptional regulatory element; Human immunodeficiency virus; Hepatitis B virus; Gene therapy; Polypurine tract; Liver; Cell culture; Rat; Digestive system; Rodentia; Nucleotide fragment; Orthohepadnavirus; Retroviridae; Experimental study; Lentivirus; Posttranscriptional modification; Virus; Vertebrata; Regulation(control); Mammalia; Hepatocyte; Hepadnaviridae; Animal; Transduction; Technique
• ISSN: 0168-8278; 1600-0641
• DOI: 10.1016/S0168-8278(01)00308-7

Background / Aims : Lentiviral vectors were designed to obtain efficient transduction of primary quiescent hepatocytes. Methods : A hepatitis B virus (HBV) fragment containing enhancers and posttranscriptional regulatory element was used to increase expression levels. The human immunodeficiency virus (HIV) central polypurine tract (PPT) was used to increase transduction of quiescent cells. HBV elements were incorporated downstream and the HIV PPT was incorporated upstream of green fluorescent protein expression cassettes in third generation self inactivating lentiviral vectors. Results : The HBV fragment increased mean fluorescence of transduced HepG2 hepatoma cells 4.3±1.7-fold and 2.3–6.0-fold in various other cell types. A role of HBV×protein in the function of the HBV element was excluded. The HBV element increased the number of transducing units per pg of HIV p24 twofold. The unmodified lentiviral vector transduced 5±1% of cultured quiescent primary rat hepatocytes, HBV elements increased transduction to 54±13% and increased fluorescence 2.8±0.6-fold. The PPT increased transduction to 47±11% and increased fluorescence 2.3±0.4-fold. The vector with PPT and HBV elements transduced 68±10% of hepatocytes and increased fluorescence synergistically, 17±6 fold. Conclusions : This study shows that HBV elements or HIV PPT are required for efficient transduction of primary hepatocytes.