Gene Transfer into Hepatocytes and Human Liver Tissue by Baculovirus Vectors

Abstract

Gene therapy of liver diseases requires the development of efficient vectors for gene transfer in vivo. Retroviral and adenoviral vectors have been shown to deliver genes efficiently into hepatocytes in vitro and in vivo. However, these vectors do not allow for exclusive infection of the liver which would be highly advantageous for in vivo gene therapy strategies. We have recently demonstrated that genetically modified baculoviruses (Autographa californica nuclear polyhedrosis virus) efficiently deliver genes into cultured cells and have a strong preference for hepatocytes of different origin. Baculoviral gene transduction efficiency into human hepatocytes was determined to approach 100% and expression levels are high, provided that gene expression is controlled by mammalian promoters. In this report, we present further properties of baculoviruses regarding their use for hepatocyte gene transfer. Baculovirus-mediated gene expression declines rapidly in the hepatocellular carcinoma cell line Huh7 and more slowly in primary cultures of mouse hepatocytes. Direct application of baculoviruses for gene delivery to the liver in vivo is hampered by serum components, presumably by complement. However, we demonstrate here that baculoviral gene transfer is feasible in ex vivo perfused human liver tissue. This result suggests the development of a strategy using baculoviral vectors for liver-directed gene therapy.

Role of paracellular junction complexes in baculovirus-mediated gene transfer to nondividing rat hepatocytes

“…11 Previous studies have reported baculovirus-mediated gene delivery to primary hepatocytes. 6,11,12 In these studies, baculovirus-mediated gene delivery was highly efficient (470%) and homogeneous. However, these studies were carried out using cells in short-term culture conditions and as such, the hepatocytes were poorly differentiated and do not have intact paracellular junctions.…”

“…56 Complement inactivation of baculoviruses can be overcome by the depletion of viral neutralizing factors in the sera prior to baculovirus infection. 12,56,57 The effects of the complement system have also been overcome by the generation of complement-resistant baculoviruses. 58 In order to test the efficacy of baculovirus vectors prior to in vivo gene therapy, an in vitro system of baculovirusmediated gene transfer with similar hepatocyte biology to the in vivo liver must be established.…”

“…2,5 The baculovirus Autographa californica multiple nucleopolyhedrovirus (AcMNPV) can efficiently deliver genes to numerous mammalian cell types including hepatic cells. [6][7][8][9][10][11][12][13][14] Recently, we have characterized baculovirusmediated gene delivery into long-term primary rat hepatocytes maintained in a chemically defined medium supplemented with DMSO. 14 Baculovirus-mediated gene transfer in this culture system was dose-dependent and restricted to peripheral cells of hepatocellular islands when extracellular calcium was present during baculovirus infection.…”

Lentiviral vectors pseudotyped with baculovirus gp64 efficiently transduce mouse cells in vivo and show tropism restriction against hematopoietic cell types in vitro

“…We demonstrated that gp64-pseudotyped lentiviral vectors efficiently transfer the human Factor IX gene or the GFP gene to mouse cells following portal vein injection, contrary to several published studies that baculovirus vectors are not infectious if injected into the vasculature of mice. 18,32 This indicates that the baculovirus infection is blocked at the postentry step and not at the receptor-binding or entry stage. The selectivity of the Ampho-pseudotyped lentiviral vectors for transduction of hepatocytes was interesting and novel.…”

“…16 Early studies showed that baculovirus vectors could efficiently transduce human hepatocyte cell lines and explanted human liver tissue. 17,18 Subsequently, it was demonstrated that baculovirus vectors could enter a broader variety of human cell types. [19][20][21] We set out to determine whether gp64 could be used to pseudotype other viral vectors and confer high infectivity in cells of hepatic origin, which are primary targets for many genetherapy applications.…”