DESCRIPTION:

FIELD OF THE INVENTION

[0001] The invention is within the scope of oncolytic virotherapy. We engineered respiratory syncytial virus (RSV) by deleting NS1 gene, and found that the NS1 gene deficient RSV (ANSI RSV) can kill breast cancer cells, but not normal human cells.

BACKGROUND OF THE INVENTION

[0002] Breast cancer: Breast cancer is the most commonly cancer among women, with more than one million new cases identified worldwide each year [1]. An estimated 192,370 patients were newly diagnosed with breast cancer in the United States in 2009, and about 40,170 died of the disease [2]. Approximately 24% to 30% of women who have no lymph-node involvement at the time of diagnosis will relapse; the relapse rate for node-positive women is between 50%-60%[3]. The 5-year survival rates for those diagnosed with regional and metastatic disease are 80% and 26%, respectively[3].

Therefore, a safe and effective treatment remains a critical need.

[0003] 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[4]. The destruction of cancer cells occurs either through direct lytic rupture by multicycle viral replication or the subsequent induction of apoptosis[5] and successful application of virotherapy requires preferential and efficient amplification of the virus to lyse cancer cells. NSl gene deficient RSV(ANS1 RSV) functions as an oncolytic virus against breast cancer.

[0004] RSV biology. RSV belongs to the family Paramyxoviridae, subfamily

Pneumovirinae, genus Pneumo virus. 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 [6]. The NSl gene, unique to members of the genus Pneumovirus [7], 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 [8]:NS1 protein is referred to as nonstructural since it has not been detected in RSV particles. NSl is exclusively found in RSV-infected cells. Our group, along with others, has found that NSl can counter the type I IFN signaling during RSV infection [9, 10], implying that NSl plays a direct role in inhibiting the host's innate immune response.

[0005] RSV can be rendered nonpathogenic by mutating the NSl gene so that it no longer inhibits IFN release, which attenuates viral infection in normal cells. However, these nonpathogenic RSV, ANSI RSV, are still oncolytic because tumor cells are defective in their ability to produce and respond to IFN and, therefore, efficiently support the propagation of ANSI RSV. SUMMARY:

[0006] This invention discloses a NS l gene deficient RSV (ANS I RSV), which could be utilize to kill breast cancer cells, but not normal human cells. In one embodiment, the gene NS l is deleted by the removal of 122 to 630 nt in the antigenomic cDNA using reverse genetics approach, resulting in the joining of the upstream nontranslated region of NS l to the translational initiation codon of NS2. The ANS I RSV was recovered through co-transfecting Vero cells with the NS 1 -deficient RSV cDNA and expressional plasmids encoding N, P, M2-1 and L. The RSV NS l protein functions as a type-I-IFN antagonist, ANS I RSV virotherapy produces more type-I-IFN, which prevents virus from replication in normal cells and also induces antitumor effects

[0007] In another embodiment, the engineered virus could be any other virus having a similar strategy to delete NS 1 gene, which functions as a gene encoding the related protein as a type-I-IFN antagonist.

[0008] In another embodiment, the ANS I RSV can be applied to cancer spot by direct injection. Or the ANS I RSV can be delivered to cancer spot through blood transfusion.

BRIEF DESCRIPTION OF THE FIGURES

[0009] Fig. 1. Diagram of the RSV genome and its transcription and replication products.

[0010] Fig. 2 A. Verify viral NS l protein by immunoblotting using anti-NS l antibodies. [001 1] Fig. 2B. Morphology of virus-infected MDA-MB-231 and CCD-1059 S cells 24h post-infection.

[0012] Fig. 2C. Viral titers as measured by plaque assay at 24h after infection. Standard deviations from three independent experiments are shown by the error bars.

[0013] Fig. 2D. In vivo test virotherapy. Subcutaneous MDA-MB-231 tumors were implanted in BALB/c nude mice and the size was photographed. Control mice received equal volume of vehicle or PBS. Tumor sizes were measured at the end of treatment. Each data point represents a mean of 6 tumors measurements plus or minus the standard deviation.

[0014] Fig. 2 E. The tumor sizes were measured, and virotherapy is indicated by arrows below the x-axis. Control mice received equal volume of vehicle or PBS. Tumor sizes were measured at the end of treatment. Each data point represents a mean of 6 tumors measurements plus or minus the standard deviation.

[0015] Fig. 2F. Viral titers were measured in different tissue homogenates from the same animal to test virus safety after three days injection of viruses.

[0016] Fig. 2G. Viral F gene expression[9] was analyzed by RT-PCR to test virus safety in different organ from the same individual after three days virotherapy.

[0017] Fig.3A. MDA-MB-231 and CCD-1059 SK cells were infected with indicated viruses (MOI = 5), and collected at 20 and 48 hr post-infection for apoptosis analysis by annexin V-binding and PI uptake assay.

[0018] Fig. 3B. MDA-MB-231 cells were infected with viruses (MOI=3) and neutralizing Abs against IFN-β (ND50 is -0.05-0.2 μg/ml PBL Interferon Source) were added 15min post-infection, and apoptosis were measured by annexin V-binding and PI uptake assay.

[0019] Fig. 3C. Vero cells were infected with indicated viruses (MOI=5) and apoptosis was measured by annexin V-binding and PI uptake assay.

[0020] Table 1. Cytopathic effect (CPE) test showing ANS I RSV selectively kills human breast cancer cells

DETAILED DESCRIPTION OF THE INVENTION

[0021 ] The respiratory syncytial virus (RSV) was used in this study. The NS l gene was deleted by the removal of 122 to 630 nt in the antigenomic cDNA using reverse genetics approach, resulting in the joining of the upstream nontranslated region of NS l to the translational initiation codon of NS2. The ANS I RSV was recovered through co- transfecting Vero cells with the NS 1 -deficient viral cDNA clone and expressional plasmids encoding N, P, M2-1 and L. Alternatively, the engineered virus could be any other viruses with the deletion of similar NS l gene.

[0022] To identify whether ANSI RSV lacks NS l gene, we infected Vero cells (IFN-β gene deficient cells) with wt RSV and ANS I RSV (MOI=5), NS l protein were tested using NS l specific antibodies by immunoblotting. As shown in Fig.2A, NS l protein was only visualized in wt RSV-infected Vero cells, not ANS I RSV-infected cells, indicating that ANS I RSV lacks NS l gene. [0023] ANS I RSV preferentially kills breast cells both in vitro and in vivo. MDA-MB- 231 breast cancer cells and normal CCD- 1059S (Human normal breast fibroblast) were cultured in as indicated by ATCC (American Type Culture Collection) instruction, and then infected with wt and ANS I RSV (MOI=5). Changes in cell morphology were observed and viral replication was measured. Fig. 2B shows that ANS I RSV selectively induces cytopathic effect (CPE) in MDA-MB-231 breast cancer cells, and that ANS I RSV has a higher viral titer in this tumor cells than in CCD-1059SK cells 24h postinfection (Fig. 2C), suggesting that MDA-MB-231 cells efficiently support the propagation of ANS I RSV. To test if ANSI RSV also kills other breast cancer cell lines, we infected breast cancer cell lines T-47D and MCF-7 with ANS I RSV (MOI=5). CPEs were observed 48 h post-infection (Table 1), indicating ANS I RSV specifically kills breast cancer cells.

[0024] To determine whether ANS I RSV infection induces tumor growth regression in vivo, MDA-MB-231 breast cancer cells were injected s.c. into the left and right flanks of 4-6 weeks old nude BALB/c mice (n=6 per group) and the resulting tumors were allowed to develop. Viruses ( 1 X 10 ¹⁰ pfu/ml) were locally injected into the tumors three times and the sizes of the tumors were measured using digital calipers. Fig. 2C, D show that ANS I RSV infection caused regression in tumor growth versus controls. To test the safety of locally administered viruses, the virus titer in various organs of infected mice was determined by plaque assay and RT-PCR assay. As shown in Fig. 2E, F, the viruses specifically localize to tumors. [0025] ANSI RSV infection induces apoptosis in tumor cells, but not in normal human breast fibroblast CCD-1059SK cells. To test the differential effect of ANS I RSV infection on apoptosis, MDA-MB-231 tumor cells and normal CCD- 1059 SK cells were infected with the indicated viruses (MOI = 5) and apoptosis was measured by the annexin V binding assay. Fig. 3A shows that ANS I RSV selectively induces apoptosis in tumor cells, compared to the cell spontaneous apoptosis shown in control.

[0026] Knockdown of the RSV NS 1 gene allows the production of more IFN-β in A549 cells [9]. To further study the involvement of IFN-β in virus-induced apoptosis in breast cancer cells, neutralizing Abs against IFN-β were used to block IFN activity, but failed to attenuate apoptosis in breast cancer cells induced by viral infection (Fig. 3B). To confirm this finding, we infected Vero cells (IFN-β gene deficient cells) with ANS I RSV, apoptosis was measured by the annexin V binding assay. Fig. 3C shows that ANS I RSV still induces apoptosis in Vero cells, compared to the control, suggesting that IFN may not be involved in virus-induced apoptosis.

Table 1. ANSI RSV selectively kills human breast cancer cells

CPE (hr post-infection)

xVirus (MOI=10) 24hr 48hr

CellsV ANSI RSV wt RSV ANSI RSV wt RSV

CCD-1059Sk (Human normal breast fibroblast) - -

MDA-MB-231 (Breast, adenocarcinoma, p53-) ++++ - +++++

T-47D (Breast, ductal carcinoma, p53-) ++ -- ++++

MCF7 (Breast, adenocarcinoma, p53+) ++ - ++++

Note: -: no CPE; ++: CPE≤ 50%; ++++: CPE >75%