Highly efficient tumor transduction and antitumor efficacy in experimental human malignant mesothelioma using replicating gibbon ape leukemia virus

Abstract

Retroviral replicating vectors (RRVs) have been shown to achieve efficient tumor transduction and enhanced therapeutic benefit in a wide variety of cancer models. Here we evaluated two different RRVs derived from amphotropic murine leukemia virus (AMLV) and gibbon ape leukemia virus (GALV), in human malignant mesothelioma cells. In vitro, both RRVs expressing the green fluorescent protein gene efficiently replicated in most mesothelioma cell lines tested, but not in normal mesothelial cells. Notably, in ACC-MESO-1 mesothelioma cells that were not permissive for AMLV-RRV, the GALV-RRV could spread efficiently in culture and in mice with subcutaneous xenografts by in vivo fluorescence imaging. Next, GALV-RRV expressing the cytosine deaminase prodrug activator gene showed efficient killing of ACC-MESO-1 cells in a prodrug 5-fluorocytosine dose-dependent manner, compared with AMLV-RRV. GALV-RRV-mediated prodrug activator gene therapy achieved significant inhibition of subcutaneous ACC-MESO-1 tumor growth in nude mice. Quantitative reverse transcription PCR demonstrated that ACC-MESO-1 cells express higher PiT-1 (GALV receptor) and lower PiT-2 (AMLV receptor) compared with normal mesothelial cells and other mesothelioma cells, presumably accounting for the distinctive finding that GALV-RRV replicates much more robustly than AMLV-RRV in these cells. These data indicate the potential utility of GALV-RRV-mediated prodrug activator gene therapy in the treatment of mesothelioma.

Figure 1.

Replication kinetics of amphotropic murine leukemia virus (AMLV) vs gibbon ape leukemia virus (GALV) vectors in human mesothelioma cells. (a) Schematic structure of AMLV-GFP and GALV-GFP vectors. These vectors contain a full-length replication-competent AMLV or GALV provirus, in which an internal ribosome entry site (IRES)-green fluorescent protein (GFP) cassette has been inserted between the env gene and 3’-untranslated region. Ψ, packaging signal; gag-pol, AMLV or GALV structural genes. (b) Replication kinetics of AMLV vs GALV vectors in human mesothelioma cells. Human non-malignant mesothelial cells (NMC and Met5A) and mesothelioma cells (H2052, H2452, MSTO and MESO1) were inoculated with AMLV-GFP or GALV-GFP vector at a multiplicity of infection of 0.01. On the days of passage, cells were analyzed for GFP expression by flow cytometry. Data are representative of three independent experiments, all yielding similar results.

Figure 2.

In vivo spread of amphotropic murine leukemia virus (AMLV) vs gibbon ape leukemia virus (GALV) vectors in malignant mesothelioma xenograft tumors. (a) In vivo fluorescence imaging. Human malignant mesothelioma MSTO tumors were grown subcutaneously in nude mice to 5–6mm in diameter, and injected intratumorally with LV-GFP (replication-deficient lentiviral vector), AMLV-GFP or GALV-GFP on day 0 (n=8 per group). At different time points indicated in the figure, whole body images (0.05- to 0.5-s exposure) were taken and analyzed by in vivo fluorescence imaging system. Representative images are shown from each group. (b) Comparison of the fluorescence intensities in MSTO tumors injected with LV-GFP, AMLV-GFP or GALV-GFP. The fluorescence intensities were normalized to the tumor volumes. Data shown are averages – (AMLV-GFP) or + (LV-GFP and GALV-GFP) s.d. from experiments (n=8 per group). (c) In vivo fluorescence imaging. Subcutaneous MESO1 tumors established in nude mice were injected intratumorally with LV-GFP, AMLV-GFP or GALV-GFP on day 0 (n=8 per group). Representative images are shown from each group. (d) Comparison of the fluorescence intensities in MESO1 tumors injected with LV-GFP, AMLV-GFP or GALV-GFP. The fluorescence intensities were normalized to the tumor volumes. Data shown are averages – (GALV-GFP) or + (LV-GFP and AMLV-GFP) s.d. from experiments (n=8 per group). D, day; W, week.

Figure 3.

Prodrug activator gene-mediated cell killing effect after amphotropic murine leukemia virus (AMLV) vs gibbon ape leukemia virus (GALV) infection in vitro. (a) Schematic structure of AMLV-CD and GALV-CD vector. These vectors were created by replacement of the internal ribosome entry site (IRES)-green fluorescent protein (GFP) cassette of AMLV-GFP and GALV-GFP with IRES-CD, respectively. CD, yeast cytosine deaminase prodrug activator gene. (b) Cell viability of Met5A, MSTO and MESO1 cells on day 18 after infection at a multiplicity of infection of 0.01 with AMLV-CD or GALV-CD. Data shown are averages ± s.d. from experiments performed in triplicate. 5FC, 5-fluorocytosine.

Figure 4.

In vivo antitumor effect of amphotropic murine leukemia virus (AMLV)- vs gibbon ape leukemia virus (GALV)-mediated prodrug activator gene therapy in subcutaneous xenograft model of human malignant mesothelioma. MESO1 tumors were grown subcutaneously in nude mice to 5–6mm in diameter, and injected intratumorally with 1×10⁴ TU (50 ml) of either AMLV-CD or GALV-CD, or phosphate-buffered saline (PBS) vehicle control on day 0, followed by intraperitoneal administration of 5-fluorocytosine (5FC; 500mgkg⁻¹ day⁻¹) every other day from day 14 to day 28 (n=10 per group). Tumor volumes were measured twice a week, and data shown are averages – (PBS and AMLV-GFP) or + (GALV-GFP) s.d. from experiments.

Figure 5.

Relative messenger RNA levels of PiT-1 and PiT-2 in cell lines by quantitative reverse transcription PCR. Total RNA was extracted from various human cells, including non-malignant human cell lines (293, fibroblast, NMC, Met5A) and malignant mesothelioma cell lines (H2052, H2452, MSTO and MESO1). The RNA was reverse-transcribed and amplified by PCR with specific primers for PiT-2, PiT-1, and glyceraldehyde 3-phosphate dehydrogenase (GAPDH). GAPDH was used as an endogenous RNA control to normalize for differences in the amount of total RNA.