Gene transfer to hepatocellular carcinoma: Transduction efficacy and transgene expression kinetics by using retroviral and lentiviral vectors

Abstract

Gene therapy is an attractive therapy for hepatocarcinoma, and several approaches have been studied using murine leukemia virus-derived retroviruses. We compared gene transfer efficacy and transgene expression kinetics after transduction of hepatocarcinoma cell lines using enhanced green fluorescent protein (EGFP)-expressing murine leukemia virus-derived retroviral vectors and HIV-derived lentiviral vectors. First, we showed that both retroviral and lentiviral vectors efficiently transduce cycling hepatocarcinoma cell lines in vitro. However, after cell cycle arrest, transduction efficacy remained the same for lentiviral vectors but it decreased by 80% for retroviral vectors. Second, we studied EGFP expression kinetics using lentiviral vectors expressing EGFP under the control of cytomegalovirus (CMV) or phosphoglycerolkinase (PGK) promoter. We show that the CMV promoter allows a stronger EGFP expression than the PGK promoter. However, in contrast to PGK-driven EGFP expression, which persists up to 2 months after transduction, CMV-driven EGFP expression rapidly decreased with time. This phenomenon is due to promoter silencing, and EGFP expression can be restored in transduced cells by using transcription activators such as interleukin-6 or phorbol myristate acetate/ionomycin and, to a lesser extent, the demethylating agent 5Ј-azacytidine. Altogether, our results suggest that lentiviral vectors, which allow efficient transduction of hepatocarcinoma cell lines with a strong and a sustained expression according to the promoter used, are promising tools for gene therapy of hepatocarcinomas.

Lentiviral vectors: optimization of packaging, transduction and gene expression

“…Although LV genomes contain an extremely low level of CpG dinucleotides [88], the ablation of these remaining sequences may favor a potential elevation of transcription. As an example, the use of SIN-derived LV vectors that do not contain the U3 LTR region has allowed sustained expression of the transgene [89,90]. A recent report has shown that the transcriptional silencing in murine cells of HIV-1-derived vectors was limited to non-SIN transfer backbones that still generate LTR-mediated transcription and therefore participate in promoter interference [91].…”

“…In addition, the choice of the ideal promoter should be seriously envisaged for optimal long-term expression performance. As an example, the benefit in terms of sustainable expression activity of the human phosphoglycerate kinase promoter (hPGK) over the cis-active promoting region of the cytomegalovirus (CMV) has been clearly demonstrated in the hepatocyte transduction model [89]. Additional heterologous promoter elements such as those derived from virus genomes have participated in an enhancement of the long-term transgene expression.…”

Gene therapy of hepatocarcinoma: a long way from the concept to the therapeutical impact

“…Lentiviral vectors are retroviral vectors, which, in some instances, have been shown to be able to transduce low-or nonproliferating cells such as neurons, nonstimulated normal hepatocytes or growth-arrested HCC cells conversely to MLV-derived vectors. [49][50][51][52][53][54][55] MLV-derived vectors and HIV-1-derived vectors share several features. The gag, pol and env coding sequences, along with the rev, vpr, nef, vif, vpu and tat coding sequences which encode regulatory and accessory proteins, have been eliminated from the vector backbone.…”

Expression of a model gene in prostate cancer cells lentivirally transduced in vitro and in vivo

“…13 Technical procedures to generate lentiviral vector containing supernatants and estimate the viral titer have already been described. 14 Transduction in vitro was performed as follows. On day 1, cells were seeded at 1 Â 10 5 cells/well in 12-well dishes in culture medium containing 4 mg/ml polybrene.…”

“…FACS analysis of EGFP expression in genetically manipulated cells harvested from tumors or metastases showed various expression profiles, suggesting that the in vivo context does indeed strongly affect the expression kinetics of these transgene. Molecular mechanisms, including DNA hypermethylation, 14,16 mutations localized in the cDNA of the transgene or in the promoter, 17,18 the impact of the cellular protein context, 19 combined with the selection of cell populations in vivo due to a potential toxic effect of the transgene in some conditions 20 can dramatically impact the expression kinetics of the transgene. Whether these mechanisms alone or in combination can explain the modification of the EGFP expression kinetics in this model remains to be explored.…”