Selden Clare, Mellor Neil, Rees Myrddin, Laurson Joanna, Kirwan Mike, Escors David, Collins Mary, Hodgson Humphrey
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.
J Gene Med. 2007 Feb;9(2):67-76. doi: 10.1002/jgm.1000.
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.
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.
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.
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.
慢病毒载体可能是转导肝细胞的首选载体；它们介导在静止细胞中的整合，并具有长期表达的潜力。在成年肝脏中，肝细胞通常处于有丝分裂静止状态。关于慢病毒载体靶细胞是否需要处于细胞周期一直存在争议；目前，人们已达成共识，即通过使用高滴度病毒可在静止肝细胞中实现有效转导。然而，降低所需感染复数（MOI）的转导方法为治疗用途提供了成本和安全性方面的潜在益处。
我们使用了两种基于HIV的新一代慢病毒载体系统（pHR-SIN-cppT SGW和pRRLSIN.cPPT.PGK.WPRE），它们编码LacZ/GFP报告基因，用于在体外转导成人和胎儿人肝细胞，同时添加或不添加生长因子、肝细胞生长因子（HGF）和表皮生长因子（EGF）。通过显微镜观察绿色荧光蛋白（GFP）的表达，并通过荧光光谱法对蛋白质表达进行定量，通过荧光激活细胞分选（FACS）分析来确定表达GFP的细胞比例，通过实时定量聚合酶链反应（PCR）来确定整合数量。
体外慢病毒转导人肝细胞后的基因表达通过生长因子HGF和EGF得到显著增强。在成年细胞中，生长因子导致更多比例的细胞每个细胞表达更多的GFP，这源于更多的整合事件。在人胎儿细胞中，转导的肝细胞比例保持不变，但细胞表达更多的GFP蛋白。
这对肝细胞基因治疗方案的设计具有启示意义，可显著降低MOI，并降低病毒介导毒性的成本和风险。
