However, serum EPO concentration returned to baseline within selleckchem Nilotinib 1 month with a peak at day 2�C4 post-transplantation. This increase in EPO was translated physiologically in a subsequent increase in hematocrit (Figure 1). Figure 1 Serum EPO and hematocrit follow-up in the three monkeys after SLIT using the mTTR-cmEPO-TK lentiviral vector. EPO, erythropoietin; SLIT, suspension with a lentiviral vector and immediately transplanted. To demonstrate that the increase in EPO was not a response to surgical bleeding, serum EPO was analyzed for its isoelectric profile. Indeed, it had been previously shown that transgene-derived EPOs following adeno-associated virus�CcmEPO vector injection in macaques presented different isoelectric patterns as compared to endogenous EPO.
12,13 Serum EPO patterns observed after SLIT were clearly different (more basic isoforms) from that of the physiological hormone (Figure 2). This demonstrates the in vivo functionality of transduced hepatocytes. Figure 2 Isoelectric patterns of serum erythropoietin (EPO). Physiological serum EPO from (A) nontransplanted monkey. Serum EPO at (B) day 2 and (C) day 8 for one monkey and (D, E) day 8 for the two other monkeys after SLIT. Cathode (?) is at the top. … Long-term survival of transduced hepatocytes and vector dissemination To determine whether transduced hepatocytes still expressed EPO in the long-term, western blot analyses were performed on liver biopsies harvested at 8 months and >1 year (day 487, day 446, and day 412, respectively) post-transplantation. EPO was detected in all liver biopsies (Figure 3b).
No EPO protein expression was detected in other tissues of transplanted macaques and in the liver of a nontransplanted macaque (Figure 3a,b). As a positive control, EPO could be detected in the kidney of nontransplanted macaques (Figure 3a). Finally, reverse transcription�CPCR analysis amplifying a region in EPO/HSV-TK expression cassette demonstrated that transgene transcription did occur in these liver biopsies (Figure 3c, lanes 1 and 2). Altogether, the data show that SLIT achieved long-term functionality of transduced hepatocytes in the liver. They also show that the designed lentiviral vector achieved a stable transduction and long-term liver-specific transgene expression in hepatocytes of macaques. Figure 3 Representative western blot and RT-PCR analysis of biopsies from macaques transplanted with transduced hepatocytes.
Western blot analysis using antibodies against EPO or tubulin, on protein extracts of (a) control nontransduced monkey liver and kidney. … To evaluate the vector biodistribution, we performed a nested Alu-long terminal repeat Cilengitide PCR analysis to detect lentiviral vector in extrahepatic tissues. Vector DNA was detected in very few extrahepatic biopsies (Figure 4). Figure 4 Detection of integrated vector provirus. Genomic DNA isolated from several tissues was subjected to nested Alu-long terminal repeat PCR.