Growth factors improve gene expression after lentiviral transduction in human adult and fetal hepatocytes.
BACKGROUND
Lentiviral vectors may be vectors of choice for transducing liver cells; they mediate integration in quiescent cells and offer potential for long-term expression. In adult liver, hepatocytes are generally mitotically 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 transduction can be achieved in quiescent hepatocytes, by using virus at high titre. 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.PGK.WPRE) encoding LacZ/GFP reporter genes to transduce adult and fetal human hepatocytes in vitro + /- growth factors, hepatocyte 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 sorting (FACS) analysis to identify the proportion of cells expressing GFP, and real-time quantitative polymerase chain reaction (PCR) for number of integrations.


RESULTS
Gene expression following lentiviral transduction of human liver cells in vitro 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 transduced 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 MOIs, and reducing both cost and risk of viral-mediated toxicity.
Selden
Clare
C
Department of Medicine, UCL Centre for Hepatology, Royal Free/Hampstead Campus, Royal Free and University College Medical School, Rowland Hill Street, Hampstead, London NW3 2PF, UK. c.selden@medsch.ucl.ac.uk
Mellor
Neil
N
Rees
Myrddin
M
Laurson
Joanna
J
Kirwan
Mike
M
Escors
David
D
Collins
Mary
M
Hodgson
Humphrey
H
D000328:Adult; D002478:Cells, Cultured; D005333:Fetus; D015870:Gene Expression / Q000187:drug effects; D005822:Genetic Vectors; D049452:Green Fluorescent Proteins / Q000378:metabolism; D006678:HIV / Q000235:genetics* / Q000378:metabolism; D017228:Hepatocyte Growth Factor / Q000494:pharmacology*; D022781:Hepatocytes / Q000166:cytology / Q000378:metabolism*; D006801:Humans; D014161:Transduction, Genetic
D049452:Green Fluorescent Proteins; D017228:Hepatocyte Growth Factor