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Transplantation of hepatocytes, whether genetically modified or not, has become an alternative to orthotopic liver transplantation for the treatment of patients with metabolic disease. However, more than ten years after the first clinical trial of ex vivo gene therapy to treat patients with Familial Hypercholesterolemia, there are still a number of impediments to these approaches. Numerous animal models are still being developed on the one hand to improve hepatocyte integration within hepatic parenchyma and function, and on the other hand to develop vectors that drive long-term transgene expression in situ. These include large animal models such as non-human primates, which have recently led to significant progress in hepatocyte transplantation. Simultaneous development of lentiviral vectors from different lentivirus species has permitted the transfer of genes into mitotically-quiescent primary cells including differentiated hepatocytes. Particularly third generation vectors derived from HIV-1 lentivirus are the most widely used and have significantly improved the safety and efficiency of these vectors. Given the shortage of organs and problems related to immunosuppression on one hand, and recent progresses in hepatocyte transduction and transplantation on the other hand, ex vivo approach is becoming a real alternative to allogeneic hepatocyte transplantation. We review the present progresses and limits of the ex vivo liver gene therapy approach in different animal models, emphasizing clinically relevant procedures.

The poor hepatocyte engraftment efficiency and the low level of their expansion in the host liver are a major limitation to cell therapy for the treatment of life-threatening liver diseases. Many rodent models have shown that liver repopulation via transplanted hepatocytes occurs only when liver growth capacity is impaired for an extended period of time. However, these models are not transposable to the clinics and to date there is no safe method to achieve this result in a clinical setting.Therefore, it is necessary to define on large animal models strategies that provide to transplanted hepatocytes sufficient proliferation stimuli to induce their division and that could permit a direct extrapolation to humans. Such procedures should be transposable to patients. We have defined a protocol of liver partial portal branch embolisation and shown that it induces the proliferation of transplanted hepatocytes in non-human primates (Macaca mulatta). This animal model is also appropriate to evaluate the lentiviral-mediated ex vivo gene therapy approach, since simian hepatocytes are efficiently transduced by HIV-1-derived lentivirus vectors.

Heterozygous mutations in JAGGED1, encoding a single-pass transmembrane ligand for the Notch receptors, cause Alagille syndrome (AGS), a polymalformative disorder affecting the liver, heart, eyes and skeleton and characterized by a peculiar facies. Most of the JAGGED1 mutations generate premature termination codons, and as a result, two pathogenic mechanisms causing AGS have been proposed: haploinsufficiency or a dominant-negative effect of putative truncated proteins. To determine whether missense or protein-truncating mutations in JAGGED1 can lead to the synthesis and function of abnormal proteins, we performed cell culture experiments. We showed that human JAGGED1 undergoes a metalloprotease-dependent cleavage resulting in the shedding of its extracellular domain and that this domain seems able to fulfill a biological function in vitro, probably by antagonizing Notch signaling. Moreover, the soluble form of JAGGED1 was able to compete with the transmembrane ligand. Mutant proteins with missense or nonsense mutations were synthesized and gave rise to a chord-like phenotype and a migration defect when expressed by stably transfected cells. These chord-like structures were similar to the phenotype exhibited by fibroblasts isolated from a fetus with a protein-truncating mutation. Results obtained from Notch signaling inhibition and Notch reporter assays showed that this chord-like phenotype, exhibited by mutant JAGGED1 transfectants, may result from an inhibitory effect on Notch signaling. Altogether, our results favor a dominant-negative mechanism of some JAGGED1 mutations in AGS.