Adult mammalian hepatocyte as target cell for retroviral gene transfer: a model for gene therapy.

Abstract

Gene therapy models have recently been developed based on the repopulation of a major organ, such as mammalian bone marrow, with cells genetically modified in vitro by infection with retroviral vectors (1-4). Another important target organ for therapeutically useful gene transfer is the liver. Unfortunately, the murine leukemia viruses (MLV) used to construct most retroviral vectors do not infect newborn, adult, or regenerating liver cells in vivo (5-8), although fetal liver can be infected and continues to express retrovitally transduced genes postnatally (7, 8). We now show that adult primary rat hepatocyte cultures (9, 10) are in fact susceptible to infection with MLV-based retroviral vectors during a period of transient dedifferentiation in vitro. Primary hepatocyte cultures exhibit many adult liver-specific functions--including gluconeogenesis and the expression of albumin, alcohol dehydrogenase, and type I pyruvate kinase--during their early lag (days 0-2) and late stationary (after day 11) phases of culture (10 16). During the intervening phase of logarithmic growth, these mature liver-specific functions are transiently but markedly decreased, and fetal hepatocyte functions such as alphal-fetoprotein and type III pyruvate kinase are expressed (12). Hepatocyte cultures were exposed at varying times during their growth cycle to a retroviral vector expressing a mutagenized but fully functional human hypoxanthine guanine phosphoribosyl transferase (HPRT) (Fig. 1, LSAPALM) (J.-K. Yee et al., in preparation). Human HPRT enzymic activity was detected in isoelectric focusing polyacrylamide gels (17) under conditions that distinguish it from the endogenous rat enzyme by its altered isoelectric point. Twoto three-dayold cultures infected with 5 x 1 0 6 infectious units of the HPRT vector exhibited human HPRT enzymic activity on day 14 postplating, as indicated by the arrows in Fig. 2A (lane a). The transduced human HPRT activity was at least 10-25% of the endogenous rat HPRT enzymic activity, equivalent to the high levels achieved in primary rat fibroblasts (Fig. 2, lane c) and human lymphoblasts (18). Since the upper bands in Fig. 2A (lanes a and c) may also contain human HPRT multimers (17), the actual transduced levels of human HPRT enzymic activity may be even higher. Figure 2B shows that human HPRT activity was present only when the hepatocyte cultures were infected during the early proliferative, dedifferentiated period of growth (days 2-3). Very low levels of human HPRT expression, not evident in Fig. 2B, were also detected on days 1 and 5, but not at any other times of infection. In addition, quiescent hepatocytes on day 1 1 can also be stimulated to reinitiate DNA synthesis and undergo additional cell division by exposure to fresh medium containing rat serum or polypeptide mitogens, mimicking the regenerating adult liver following partial hepatectomy (9, 19).