Marilyn Fernandez

Genetically Engineered Vesicular Stomatitis Virus in Gene Therapy: Application for Treatment of Malignant Disease

ABSTRACT We report here the generation of recombinant vesicular stomatitis virus (VSV) able to produce the suicide gene product thymidine kinase (TK) or cytokine interleukin 4 (IL-4). In vitro cells infected with the engineered viruses expressed remarkably high levels of biologically active TK or IL-4 and showed no defects in replication compared to the wild-type virus. Recombinant viruses retained their ability to induce potent apoptosis in a variety of cancer cells, while normal cells were evidently more resistant to infection and were completely protected by interferon. Significantly, following direct intratumoral inoculation, VSV expressing either TK or IL-4 exhibited considerably more oncolytic activity against syngeneic breast or melanoma tumors in murine models than did the wild-type virus or control recombinant viruses expressing green fluorescent protein (GFP). Complete regression of a number of tumors was achieved, and increased granulocyte-infiltrating activity with concomitant, antitumor cytotoxic T-cell responses was observed. Aside from discovering greater oncolytic activity following direct intratumoral inoculation, however, we also established that VSV expressing IL-4 or TK, but not GFP, was able to exert enhanced antitumor activity against metastatic disease. Following intravenous administration of the recombinant viruses, immunocompetent BALB/c mice inoculated with mammary adenocarcinoma exhibited prolonged survival against lethal lung metastasis. Our data demonstrate the validity of developing novel types of engineered VSV for recombinant protein production and as a gene therapy vector for the treatment of malignant and other disease.

Development of Recombinant Vesicular Stomatitis Viruses That Exploit Defects in Host Defense To Augment Specific Oncolytic Activity

ABSTRACT Vesicular stomatitis virus (VSV) is a negative-stranded RNA virus normally sensitive to the antiviral actions of alpha/beta interferon (IFN-α/β). Recently, we reported that VSV replicates to high levels in many transformed cells due, in part, to susceptible cells harboring defects in the IFN system. These observations were exploited to demonstrate that VSV can be used as a viral oncolytic agent to eradicate malignant cells in vivo while leaving normal tissue relatively unaffected. To attempt to improve the specificity and efficacy of this system as a potential tool in gene therapy and against malignant disease, we have genetically engineered VSV that expresses the murine IFN-β gene. The resultant virus (VSV-IFNβ) was successfully propagated in cells not receptive to murine IFN-α/β and expressed high levels of functional heterologous IFN-β. In normal murine embryonic fibroblasts (MEFs), the growth of VSV-IFNβ was greatly reduced and diminished cytopathic effect was observed due to the production of recombinant IFN-β, which by functioning in a manner involving autocrine and paracrine mechanisms induced an antiviral effect, preventing virus growth. However, VSV-IFNβ grew to high levels and induced the rapid apoptosis of transformed cells due to defective IFN pathways being prevalent and thus unable to initiate proficient IFN-mediated host defense. Importantly, VSV expressing the human IFN-β gene (VSV-hIFNβ) behaved comparably and, while nonlytic to normal human cells, readily killed their malignant counterparts. Similar to our in vitro observations, following intravenous and intranasal inoculation in mice, recombinant VSV (rVSV)-IFNβ was also significantly attenuated compared to wild-type VSV or rVSV expressing green fluorescent protein. However, VSV-IFNβ retained propitious oncolytic activity against metastatic lung disease in immunocompetent animals and was able to generate robust antitumor T-cell responses. Our data indicate that rVSV designed to exploit defects in mechanisms of host defense can provide the basis for new generations of effective, specific, and safer viral vectors for the treatment of malignant and other disease.

NFAR-1 and -2 modulate translation and are required for efficient host defense

We report here that the alternatively spliced nuclear factors associated with double-stranded RNA, NFAR-1 (90 kDa) and -2 (110 kDa), are involved in retaining cellular transcripts in intranuclear foci and can regulate the export of mRNA to the cytoplasm. Furthermore, the NFAR proteins were found to remain associated with exported ribonucleoprotein complexes. Loss of NFAR function, which was embryonic-lethal, caused an increase in protein synthesis rates, an effect augmented by the presence of the mRNA export factors TAP, p15, or Rae1. Significantly, NFAR depletion in normal murine fibroblasts rendered these cells dramatically susceptible to vesicular stomatitis virus replication. Collectively, our data demonstrate that the NFARs exert influence on mRNA trafficking and the modulation of translation rates and may constitute an innate immune translational surveillance mechanism important in host defense countermeasures against virus infection.

Arabinosylcytosine downregulates thymidine kinase and induces cross-resistance to zidovudine in T-lymphoid cells.

The aim of this study was to determine molecular mechanism(s) responsible for the reduced thymidine kinase activity (TK) observed earlier in an arabinosylcytosine (araC) resistant lymphoid cell line (H9-araC cells), which was obtained following continuous cultivation of H9 cells in the presence of 0.5 microM araC. Compared to H9 cells, in H9-araC cells TK1 and TK2 gene expressions were reduced to 17.7% and 2.5%, respectively, and the cellular AZT accumulation was diminished to 35.8%. These cells were also found cross-resistant to azidothymidine (>42-fold). There was no significant difference in the expression of MDR1, MRP4 or TK protein. The lack of correlation between the expressions of TK protein and TK1 and TK2 suggests that post-translational factors may also play a role in the reduced TK activity in H9-araC cells. These findings suggest that araC affects TK expression at the genetic level.