Melanoma: The incidence of melanoma is increasing worldwide. Despite decades of clinical research, patients with advanced melanoma continue to have a poor prognosis, and no agents have shown statistically significant improvement in overall survival in the patients with metastatic melanoma [1]. For patients with surgically resected, thick (≧2 mm) primary melanoma with or without regional lymph node metastases, the only effective adjuvant therapy is type I interferon [2]. Standard recommended therapy for patients with metastasis is single-agent dacarbazine, but responses to this agent and its oral analogue, temozolomide, are <15% and generally transient [3]. Biochemotherapy increases objective response rates but has not been shown to significantly improve survival compared with chemotherapy alone and is associated with additive toxicity [4]. Therefore, a safe and effective treatment remains a critical need.
Oncolytic virotherapy. Oncolytic virotherapy is a novel strategy using viruses, either naturally occurring or genetically modified, to selectively target and destroy tumor cells whilst leaving surrounding non-malignant cells unharmed [5]. The destruction of cancer cells occurs either through direct lytic rupture by multi-cycle viral replication or the subsequent induction of apoptosis [6] and successful application of virotherapy requires preferential and efficient amplification of the virus to lyse cancer cells. NS1/NS2 gene deficient RSV (ΔNS1/NS2 RSV) functions as an oncolytic virus killing melanoma tumor cells.
RSV biology. RSV belongs to the family Paramyxoviridae, subfamily Pneumovirinae, genus Pneumovirus. The viral RNA is approximately 15 kb in size and is flanked by a leader region at the 3′ extremity of the genome and by a trailer region at the 5′ extremity (FIG. 1). The viral genome contains individual genes for ten viral proteins [7]. The NS1 gene, unique to members of the genus Pneumovirus [8], is promoter-proximally located at the 3′ end of the viral genome and its mRNA is the most abundant of the RSV transcripts in a linear start-stop-restart mode [9]. NS1 and NS2 are referred to as nonstructural since they have not been detected in RSV particles. NS1 and NS2 are exclusively found in RSV-infected cells. Our group, along with others, has found that NS1 and NS2 can counter the type I IFN signaling during RSV infection [10, 11], implying that NS1 and NS2 play an important role in inhibiting the host's innate immune response.
RSV can be rendered nonpathogenic by mutating the NS1 and NS2 genes so that it no longer inhibits IFN release (FIG. 2), which attenuates viral infection in normal cells. However, these nonpathogenic RSV, ΔNS1/NS2 RSV, are still oncolytic (FIG. 3A) because tumor cells are defective in their ability to produce and respond to IFN and, therefore, efficiently support the propagation of ΔNS1/NS2 RSV. ΔNS1/NS2 RSV replicates to a high titer in melanoma cancer cells, compared to the normal primary epidermal melanocytes (FIG. 3B), and ΔNS1/NS2 RSV, not wt RSV, specifically kills melanoma cancer cells, but not normal human primary epidermal melanocytes PCS-200-013 cells (Table 1).
ΔNSI/NS2 RSV Induces Apoptosis in Human Melanoma Cancer Cells.
Evasion from apoptotic cell death unregulated cell proliferation and eventual tumor development is one of the hallmarks of oncogenic cell-transformation. We found that ΔNS1/NS2 RSV selectively induces apoptosis in tumor cells (FIG. 4A-B), and also generated cytopatheic effect (CPE) in melanoma cancer cells (FIG. 3A) as we demonstrated in our previous patent application U.S. 61/398,236, suggesting that multiply mechanism-mediated cell death participates in the anti-tumor effect of ΔNS1/NS2 RSV.
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