Construction of a replication-competent retroviral vector for expression of the VSV-G envelope glycoprotein for cancer gene therapy.

Gibbon ape leukemia virus (GALV) can infect a wide variety of cells but fails to infect most cells derived from laboratory mice. Transduction of human hematopoietic stem cells with GALV retroviral vectors is more efficient than with amphotropic vectors. In this study, a Moloney murine leukemia virus-gibbon ape leukemia virus (MoMLV-GALV) vector was constructed by replacing the natural env gene of the full-length Moloney MLV genome with the GALV env gene. To monitor viral transmission by green fluorescent protein (GFP) expression, internal ribosomal entry site-enhanced GFP (IRES-EGFP) was positioned between the GALV env gene and the 3' untranslated region (3' UTR) to obtain pMoMLV-GALV-EGFP. The MoMLV-GALV-EGFP vector was able to replicate with high titer in TE671 human rhabdomyosarcoma cells and U-87 human glioma cells. To evaluate the potential of the MoMLV-GALV vector as a therapeutic agent, the gene for the fusogenic envelope G glycoprotein of vesicular stomatitis virus (VSV-G) was incorporated into the vector. Infection with the resulting MoMLV-GALV-VSV-G vector resulted in lysis of the U-87 cells due to syncytium formation. Syncytium formation was also observed in the transfected human prostate cancer cell line LNCaP after extended cultivation of cells. In addition, we deleted the GALV env gene from the MoMLV-GALV-VSV-G vector to improve viral genome stability. This MoMLV-VSV-G vector is also replication competent and induces syncytium formation in 293T, HT1080, TE671 and U-87 cells. These results suggest that replication of the MoMLV-GALV-VSV-G vector or MoMLV-VSV-G vector may directly lead to cytotoxicity. Therefore, the vectors developed in this study are potentially useful tools for cancer gene therapy.

Construction of replication-competent oncolytic retroviral vectors expressing R peptide-truncated 10A1 envelope glycoprotein.

Replication-deficient retroviral (RDR) vectors have been generally used for gene therapy, but clinically beneficial transduction efficiency is difficult to achieve with these vectors. In recent times, attention has been focused on the use of murine leukemia virus (MLV)-based replication-competent retroviral (RCR) vectors. RCR vectors have been shown to achieve efficient tumor reduction in a wide variety of cancer models. Most RCR vectors have been developed from amphotropic 4070 A MLV env, which is broadly applied in basic research. In this study, we generated RCR vectors based on Moloney MLV by replacing the native env gene in a full-length viral genome with the 10A1 env gene. 10A1 MLV can infect a wide variety of cells. Unlike amphotropic MLV, the 10A1 MLV can use amphotropic MLV receptor Pit2 or gibbon ape leukemia virus (GaLV) receptor Pit1. The resulting construct MoMLV-10A1-EGFP was able to replicate in 293 T, NIH3T3, and Mus dunni cells. To evaluate the potential of MoMLV-10A1 vector as a therapeutic agent, we incorporated the yeast cytosine deaminase (CD) suicide gene into vectors. The resulting vector MoMLV-10A1-CD could inhibit the growth of human 293T cells upon 5-fluorocytosine (5-FC) administration. In addition, to lyse tumor cells by syncytium, MoMLV-10A1-R(-)-EGFP was generated by replacing wild-type 10A1 env with the 16-amino acid R peptide-truncated 10A1 env gene. Syncytium formation was observed in the TE671 human tumor cells, 293 T and PG13 cells upon transfection of the MoMLV-10A1-R(-)-EGFP vector. This result suggests that replication of this vector could be oncolytic in itself. We also found that syncytium could contribute to enhance cell-to-cell transmission of the retroviral vectors. Our results thus show that the MoMLV-10A1 vectors can be potentially useful for cancer gene therapy.

Human-APOBEC3G-dependent restriction of porcine endogenous retrovirus replication is mediated by cytidine deamination and inhibition of DNA strand transfer during reverse transcription.

Although human apolipoprotein B mRNA-editing enzyme catalytic polypeptide-like 3G (APOBEC3G, hA3G)-mediated deamination is the major mechanism used to restrict the infectivity of a broad range of retroviruses, it is unclear whether porcine endogenous retrovirus (PERV) is affected by hA3G or porcine A3F (poA3F). To determine whether DNA deamination is required for hA3G- and poA3F-dependent inhibition of PERV transmission, we developed VSV-pseudotype PERV-B expressing hA3G, mutant hA3G-E67Q (encapsidation and RNA binding activity-deficient), mutant hA3G-E259Q (deaminase-deficient), or poA3F. hA3G-E67Q decreased virus infectivity by ~ 93% compared to the ~ 99% decrease of viral infectivity by wild-type hA3G, while hA3G-E259Q decreased the infectivity of PERV-B by ~ 35%. These data suggest that cytidine deamination activity is crucial for efficient restriction of PERV by hA3G, but cytidine deamination cannot fully explain the inactivation of PERV by hA3G. Furthermore, differential DNA denaturation PCR (3D-PCR) products from 293T cells infected with PERV-B expressing hA3G mutants were sequenced. G-to-A hypermutation was detected at a frequency of 4.1% for hA3G, 3.4% for hA3G-E67Q, and 4.7% for poA3F. These results also suggest that hA3G and poA3F inhibit PERV by a deamination-dependent mechanism. To examine the effect of hA3G on the production of PERV DNA, genomic DNA was extracted from 293T cells 12 h after infection with PERV expressing hA3G, and this DNA was used as template for real-time PCR. A 50% decrease in minus strand strong stop (-sss) DNA synthesis/transfer was observed in the presence of hA3G. Based on these results, we conclude that hA3G may restrict PERV by both deamination-dependent mechanisms and inhibition of DNA strand transfer during PERV reverse transcription.