723. VSV-G Pseudotyped, Semi-Replication Competent MLV Retroviruses for Cancer Gene Therapy
Authors: Jian Qiao, Jesus Moreno, Timothy Kottke, Michael Forshaw, Rosa Maria Diaz, Richard Vile
Journal: Molecular Therapy (2005)

Low levels of gene delivery in vivo using non-replicating retroviral vectors has severely limited their application for clinical protocols. Therefore, we have investigated the potential of replication-competent retroviruses for cancer gene therapy. To alleviate safety concerns, and to increase the payload capacity, gagpol and env were encoded separately on two different vectors. We used the VSV-G env to overcome problems associated with interference of viral superinfection when infecting the same cell with the two different gag-pol and env viruses, a system we have termed as a semi-replication-competent retrovirus. This also has the added advantage that the VSV-G protein will serve a dual purpose as both pseudotyping viral envelope and therapeutic protein. Semi-replicative vectors were generated with at least two log higher titres than classical, VSV-G pseudotyped, replication defective vectors, and both replicated and spread through cultured cells to high levels. In addition, these semi-replicative vectors induced tumor killing during viral spread. This tumor cytotoxicity was significantly improved by incorporating the HSV-tk suicide gene into one of the vectors in combination with GCV treatment. Fast growing human xenografts of human fibrosarcoma HT1080 cells, pre-infected by a semi-replicative GFP vector pair, abrogated tumorigenicity completely in athymic nu/nu mice. At a ratio of 1:1 of pre-infected cells with parental cells, the semi-replicative HSVtk pair of viruses was able to eradicate all tumor growth and survival was still significantly prolonged with the semi-replicative GFP vector pair. More importantly, direct intratumoral injection of the semi-replicative HSVtk virus pair, followed by GCV treatment, led to reductions in tumor size of up to 70% of established subcutaneous HT1080 tumors compared to the untreated groups (111.63 + 11.11 mm3 vs. 377.50 + 143.92 mm3). When the same semi-replicative HSVtk virus pairs were injected intratumorally into established B16 murine melanoma growing in immune-competent C57Bl/6mice, significantly prolonged survival rate was obtained compared to control injected tumors. To further improve the efficacy of this therapy, we injected autologous or xenogeneic cells producing the semi-replicative viruses directly into established B16 tumors. Expression of the HSVtk semi replicative virus pair, along with the added immune adjuvant effects of xenogeneic producer cells, led to further survival advantages compared to other groups of treatments. We were unable to detect viral integration in a range of normal organs by PCR. There was also no evidence of recombination of the two vectors either in vitro or in vivo by Southern blotting of genomic DNA extracted from infected tumor cells. These data show that direct tumor cell cytotoxicity can be provided by use of semi replicating retroviruses at greatly improved levels compared to replication defective vectors; moreover, a combination of suicide gene therapy delivered by semi-replication competent vectors, with concomitant immune potentiation, can lead to highly significant survival advantages.