Hepatocytes are known to be in a quiescent status, and the toxic triphosphate processes by the suicide gene HSV-TK after selleck screening library GCV administration theoretically need cell replication to induce apoptosis. The fact that administration of GCV results in depletion of EPO/TK�Cexpressing hepatocytes is probably due to the incorporation of the toxic triphosphates into mitochondrial DNA.30 Braun and colleagues also reported that HSV-TK�Cexpressing hepatocytes were ablated in adult animals in HSV-TK transgenic mice upon GCV treatment.31 A low systemic dissemination of the vector in extrahepatic tissues was pointed out by Alu-long terminal repeat PCR. Alu-long terminal repeat PCR was specifically used to distinguish the integrated provirus from the plasmidic contamination (data not shown).

Dissemination is probably due to noninternalized lentiviral vectors that adhere to hepatocyte membranes resulting in in vivo lentiviral vector spreading.32 Because brain biopsies were taken at the time of killing, i.e., after GCV-mediated ablation of transduced hepatocytes, the brain-detected vector DNA may be due to monocytes/macrophages trafficked into brain after having phagocyted apoptotic transduced hepatocytes and/or being lentivirally transduced.33,34,35 We observed that the vector integration in nonhepatic tissues does not lead to protein expression. This demonstrates the liver specificity of mTTR promoter in nonhuman primates and suggests inactivity of mTTR enhancers in these tissues. This inactivity may be important considering that the in vivo genotoxicity side effects reported for oncoretroviral vectors were mostly related to retroviral enhancer activity.

36 No gross macroscopic or histological anomalies were detected in any organ at the killing of macaques. Nevertheless, because it is important to avoid vector dissemination, we performed preliminary experiments to completely remove noninternalized infectious virus after transduction. Our preliminary results show that the sequential incubation of transduced hepatocytes with a protease Batimastat and human serum efficiently eliminated those infectious virus particles without impairing viability of transduced hepatocytes (J. Birraux, B.E. Wildhaber, C. Jond, D.C. Belli, and O. Menzel, personal communication). Protease treatment would release cell surface�Cbound viruses and human serum was shown to inactivate vesicular stomatitis virus glycoprotein�Cpseudotyped lentiviral vectors.37 In conclusion, this study demonstrates the feasibility of SLIT in the Macaca fascicularis, an animal model very close to human infants, with long-term (more than a year) survival and functionality of the autotransplanted lentivirally transduced hepatocytes.