Engraftment of Autologous Retrovirally Transduced Hepatocytes after Intraportal Transplantation into Nonhuman Primates: Implication forex VivoGene Therapy

Abstract

The main impediment to effective ex vivo liver gene therapy of metabolic diseases is the lack of experimental work on large animals to resolve such important issues as effective gene delivery, cell-processing techniques, and the development of appropriate vectors. We have used a nonhuman primate, as a preclinical model, to analyze the limiting steps of this approach using recombinant retroviruses. Seven monkeys (Macaca fascicularis) underwent the complete protocol: their left liver lobe was resected, a catheter was placed in the inferior mesenteric vein and connected to an infusion chamber, and the hepatocytes were isolated, cultured, and transduced with a retroviral vector containing the beta-galactosidase gene. The hepatocytes were harvested and returned to the host via the infusion chamber. Biopsies were taken 4-40 days later. No animal was killed in the course of the experiments. They all tolerated the procedure well. We have developed and defined conditions that permit the proliferation and transduction of up to 90% of the plated hepatocytes. A significant proportion of genetically modified cells, representing up to 3% of the liver mass, were safely delivered to the liver via the chamber. Polymerase chain reaction analysis detected integrated viral DNA sequences and quantitative analysis of the in situ beta-Gal-expressing hepatocytes indicated that a significant amount of transduced hepatocytes, up to 2%, had become integrated into the liver and were functional. These results represent substantial advances in the development of the ex vivo approach and suggest that this approach is of clinical relevance for liver-directed gene therapy.

A highly efficient, stable, and rapid approach for ex vivo human liver gene therapy via a FLAP lentiviral vector

“…Recent improvements in oncoretroviral vector titers enabled efficient gene transfer in plated simian hepatocytes using multiple rounds of transduction before reintroduction into the animal. 22 The development of lentiviral vectors seems to have introduced a new major breakthrough in both in vivo and ex vivo liver gene therapy. 23,24 We show herewith that, using a lentiviral vector, we can bypass the critical steps of the hepatocyte ex vivo transduction procedure, namely, the plating and further deleterious trypsin dissociation of cultured hepatocytes.…”

Improved gene transfer selectivity to hepatocarcinoma cells by retrovirus vector displaying single-chain variable fragment antibody against c-Met

“…This inability to readily infect human hepatocytes was also observed with monkey hepatocytes; 1% of these cells were infected with the 3D6-Env virus, whereas up to 90% of these cells were infected with the wild-type envelope virus. 35 To determine whether the partial inhibition of the 3D6-Env virus observed on c-Met-expressing cells was due to its binding to the c-Met receptor, HuH7 were preincubated with increasing amounts of 3D6 antibodies to saturate c-Met before infection. This resulted in increased infectivity, which was correlated to the amount of 3D6 antibodies added onto the cells (Figure 5c).…”

Improved Survival of Fulminant Liver Failure by Transplantation of Microencapsulated Cryopreserved Porcine Hepatocytes in Mice

“…Recently, porcine hepatocytes have been used clinically in bioartificial livers (BAL) (13). As immune and several approaches are currently tested: (i) extracorporeal hybrid bioartificial liver devices (22,28), (ii) imresponses are still major barriers for successful cell and organ xenotransplantation, semipermeable capsules usplantable bioartificial liver devices (6,9,10), and (iii) hepatocyte transplantation (1,7,8,16,17,21,36,37,39).…”

“…The small pores of the capsules prevent Hepatocyte allotransplantation has been used successfully in rodent (16,21,37) and primates (1,39) as therapy cells and antibodies to cross, while oxygen, glucose, and nutrients can pass through the membranes and maintain of chronic or acute liver failure. In humans, studies showed that hepatocyte allotransplantation was useful metabolic functions.…”