- view fewer
Growth factors improve gene expression after lentiviral transduction in human adult and fetal hepatocytes.
Journal of Gene Medicine
67 - 76.
Background: Lentiviral vectors may be vectors of choice for transducing liver cells; they mediate integration in quiescent cells and offer potential for tong-term expression. In adult liver, heparocytes are generally mitorically quiescent. There has been controversy as to the necessity for lentiviral vector target cells to be in the cell cycle; currently, there is consensus that effective tranduction can be achieved in quiescent hepatocytes, by using virus at high time. However, transduction approaches which reduce the multiplicities of infection (MOIs) required provide potential benefit of cost and safety for therapeutic use. Methods: We used two late-generation HIV-based lentiviral vector systems (pHR SIN-cppT SGW and pRRLSIN.cPPT.PGF-WPRE) encoding LaeZ/GFP reporter genes to transduce adult and fetal human hepatocytes in vitro +/- growth factors, heparocyte growth factor (HGF) and epidermal growth factor (EGF). Green fluorescent protein (GFP) expression was observed microscopically, and quantified by fluorescence spectrometry for protein expression, fluorescence-activated cell setting (FACS) analysis to identify the proportion of cells expressing GFP, and real-time quantitative potymerase chain reaction (PCR) for number of integrations. Results: Gene expression following lentiviral transduction of human liver cells in vivo was markedly enhanced by the growth factors HGF and EGF. In adult cells growth factors led to a greater proportion of cells expressing more GFP per cell, from more integration events. In human fetal cells, the proportion of transcduced hepatocytes remained identical, but cells expressed more GFP protein. Conclusions: This has implications for the design of regimes for liver cell gene therapy, allowing marked reduction of MOls, and reducing both cost and risk of viral-mediated toxicity. Copyright © 2007 John Wiley & Sons, Ltd.
|Title:||Growth factors improve gene expression after lentiviral transduction in human adult and fetal hepatocytes|
Archive Staff Only