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, 18 (2), 275-84

Engineered Newcastle Disease Virus as an Improved Oncolytic Agent Against Hepatocellular Carcinoma

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Engineered Newcastle Disease Virus as an Improved Oncolytic Agent Against Hepatocellular Carcinoma

Jennifer Altomonte et al. Mol Ther.

Abstract

Newcastle disease virus (NDV) is an intrinsically tumor-specific virus, which is currently under investigation as a clinical oncolytic agent. Several clinical trials have reported NDV to be a safe and effective agent for cancer therapy; however, there remains a clear need for improvement in therapeutic outcome. The endogenous NDV fusion (F) protein directs membrane fusion, which is required for virus entry and cell-cell fusion. Here, we report a novel NDV vector harboring an L289A mutation within the F gene, which resulted in enhanced fusion and cytotoxicity of hepatocellular carcinoma (HCC) cells in vitro, as compared with the rNDV/F3aa control virus. In vivo administration of the recombinant vector, termed rNDV/F3aa(L289A), via hepatic arterial infusion in immune-competent Buffalo rats bearing multifocal, orthotopic liver tumors resulted in tumor-specific syncytia formation and necrosis, with no evidence of toxicity to the neighboring hepatic parenchyma. Furthermore, the improved oncolysis conferred by the L289A mutation translated to significantly prolonged survival compared with control NDV. Taken together, rNDV/F(L289A) represents a safe, yet more effective vector than wild-type NDV for the treatment of HCC, making it an ideal candidate for clinical application in HCC patients.

Figures

<b>Figure 1</b>
Figure 1
rNDV/F3aa(L289A) is sensitive to the inhibitory effects of interferon (IFN). (a) Primary human hepatocytes (PHHs), two human hepatocellular carcinoma (HCC) cell lines [(b) Huh7 and (c) HepG2], and (d) a rat HCC cell line (McA-RH7777) were untreated or treated overnight with increasing concentrations of universal type I interferon (IFN) (10–1,000 IU/ml) in 24-well dishes. Triplicate wells were infected with rVSV-LacZ, rNDV/F3aa, or rNDV/F3aa(L289A) at a multiplicity of infection of 1. Viral titers were determined by TCID50 of conditioned media harvested 24 hours postinfection. Values represent the mean ± SD. NDV, Newcastle disease virus; TCID50, 50% tissue culture infectious dose; VSV, vesicular stomatitis virus.
<b>Figure 2</b>
Figure 2
rNDV/F3aa(L289A) induces interferon (IFN) signaling in primary human hepatocytes (PHHs). PHHs, two human hepatocellular carcinoma (HCC) cell lines (Huh7 and HepG2), and a rat HCC cell line (McA-RH7777) were co-transfected with (a) pIFN-β-Luciferase or (b) pISRE-Luciferase and pRL-Luciferase in 24-well dishes. At 24 hours post-transfection, cells were mock-treated, stimulated with polyinosinic:polycytidylic acid (pI:C) (2.5 mg/ml) or IFN (1,000 IU/ml), or infected with rVSV-LacZ, rNDV/F3aa, or rNDV/F3aa(L289A) at a multiplicity of infection of 1.0 and incubated overnight. Firefly-luciferase activity was normalized to renilla activity to control for transfection efficiency. Fold-induction of the promoters was calculated as a function of luciferase activity in stimulated versus mock-treated cells. Data are presented as the mean ± SD of triplicate measurements. NDV, Newcastle disease virus; VSV, vesicular stomatitis virus.
<b>Figure 3</b>
Figure 3
F(L289A) modification results in enhanced fusogenic activity of Newcastle disease virus (NDV). Human (Huh7 and HepG2) and rat (McA-RH7777) cells were mock infected or infected with rNDV/F3aa or rNDV/F3aa(L289A) at a multiplicity of infection of 0.1. At 48 hours postinfection, cells (a) were stained for β-gal expression or (b) analyzed for syncytial index. The syncytial index was calculated as the number of nuclei per syncytia divided by the number of nuclei per field of view. Each cell line was analyzed in triplicate, and data are expressed as the mean ± SD.
<b>Figure 4</b>
Figure 4
NDV causes transient body weight loss and elevation of liver enzymes in rats. Male non-tumor-bearing Buffalo rats were treated with PBS, or 108 TCID50 of rNDV/F3aa or rNDV/F3aa(L289A) by hepatic arterial infusion (N = 5). (a) Body weight was recorded daily, and (b) blood was drawn at the indicated time points for measurement of serum chemistries (right panel). Values are expressed as mean ± SD. BUN, blood urea nitrogen; GOT, glutamic oxaloacetic transaminase; GPT, glutamic pyruvic transaminase; NDV, Newcastle disease virus; PBS, phosphate-buffered saline; TCID50, 50% tissue culture infectious dose.
<b>Figure 5</b>
Figure 5
NDV/F3aa(L289A) treatment results in tumor-specific necrosis and virus replication in hepatocellular carcinoma (HCC)–bearing rats. Male Buffalo rats bearing multifocal orthotopic HCC nodules were treated with rNDV/F3aa(L289A) (108 TCID50) by hepatic arterial infusion, and killed at the indicated time points post-treatment (n = 3). (a) Tumor-containing liver sections were subjected to hematoxylin and eosin staining and analyzed for tumor necrosis and liver toxicity. Representative sections are shown at ×5 (overview) and ×20 (magnification of tumor and liver sections) magnification. Morphometric analysis of tumor necrosis was performed using ImageJ software (National Institutes of Health), and is shown as the mean ± standard deviation at each time point. (b) Tumor-containing liver sections were subjected to β-gal staining. Representative sections are shown at ×5 (overview) and ×20 (magnification of tumor and liver sections) magnification. Viral titers were quantified by TCID50 analysis of liver and tumor lysate, and are expressed as the mean ± SD. NDV, Newcastle disease virus; TCID50, 50% tissue culture infectious dose.
<b>Figure 6</b>
Figure 6
Infiltration of natural killer (NK) and myeloperoxidase (MPO) cells to NDV/F3aa(L289A) infected hepatocellular carcinoma (HCC) tumors. Rats bearing multifocal HCC lesions were treated by transarterial infusion of NDV/F3aa(L289A) and killed at 30 minutes, and day 1, 3, 5, and 7 after treatment. Tumor-containing liver sections were processed for immunohistochemical staining for (a) NK cell marker and (b) MPO. Positive stained cells were quantified using ImageJ software (NIH), and the mean ± SD are shown. Representative immunohistochemical sections are presented in (c) ×5 and (d) ×20 magnification. NDV, Newcastle disease virus.
<b>Figure 7</b>
Figure 7
rNDV/F3aa(L289A) therapy results in significant survival prolongation hepatocellular carcinoma (HCC)–bearing rats. Male Buffalo rats bearing multifocal HCC lesions were treated by hepatic arterial infusion of phosphate-buffered saline (PBS) (n = 10), rNDV/F3aa (n = 14), or rNDV/F3aa(L289A) (n = 13) and followed for survival. Animals were monitored daily, and survival was plotted as a Kaplan–Meier survival curve. P values for PBS versus rNDV/F3aa is <0.02, for rNDV/F3aa versus rNDV/F3aa(L289A) is <0.04. Statistical significance was determined by log-rank test. NDV, Newcastle disease virus.

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