Abstract
Recombinant Newcastle disease virus (rNDV) is tumor selective and intrinsically oncolytic, which has been developed as a vector to express exogenous genes to enhance its oncolytic efficacy. Our previous studies found that insertion sites of foreign gene in rNDV vector affected its expression and anti-tumor activities. However, the optimal insertion site for foreign genes remains unknown. In this study, we inserted the enhanced green fluorescence protein (EGFP) and IL2 genes into four different intergenic regions of the rNDV using reverse genetics technology. Recombinants rNDV-EGFPs and rNDV-IL2s were successfully rescued, which displayed the similar growth kinetics with parental virus. Both EGFP mRNA and protein levels were most abundant in HepG2 cells, when EGFP gene was inserted between the NP/P site of the rNDV. Similarly, the IL-2 expressed by HepG2 cells infected with rNDV-IL2 was highest, when IL2 was inserted into NP/P site. To test whether these rNDVs that express higher foreign genes could induce stronger anti-tumor response, we treated the H22-oxter-tumor-bearing C57BL/6J mice with rNDV-IL2s and then examined the oncolytic efficacy. The results showed that rNDV-IL2-NP/P had the strongest inhibition of murine hepatoma carcinoma tumors. The splenocytes isolated from the mice treated with rNDV-IL2-NP/P reached the highest degrees of CD4+ T and CD8+ T cells. In addition, animals’ survival rate in rNDV-IL2-NP/P-treated group was higher than that of other groups. Taken together, these results demonstrate that NP and P gene junction in rNDV is the optimal insertion site for foreign genes expression to enhance rNDV’s anti-tumor effects.
Highlights
Newcastle disease virus (NDV), a single negative stranded RNA virus, belongs to member of the Avulavirus genus in the Paramyxoviridae family
This is the first study to demonstrate that the non-coding region between the NP and P gene junction is an optimal insertion site for foreign gene expression to enhance the antitumor effects of Recombinant Newcastle Disease Virus (rNDV)
Growth kinetics of the rNDVs was not distinguishable from that of the parent virus, which indicated that the insertion of the enhanced green fluorescence protein (EGFP) or IL2 in the genome had less effect on the viral life cycles
Summary
Newcastle disease virus (NDV), a single negative stranded RNA virus, belongs to member of the Avulavirus genus in the Paramyxoviridae family. The NDV genome contains six transcriptional units (NP, P, M, F, HN and L), which encode eight proteins in the order NP-P/V/ I-M-F-HN-L [1]. The genome of NDV contains a series of non-transcribed intergenic (IG) sequences. These junction regions consist of three elements, which are known as gene-end (GE), IG, and gene-start (GS) sequences [2]. From the 3’ end to the 5’ end, NDV genome is transcribed by a stop-and-restart mechanism at each transcriptional unit, and the expression levels of viral proteins are attenuated from the NP to L protein [3]. Clinical trials for cancer treatment have demonstrated NDV to be well tolerated with few adverse side effects [6]. These events make NDV a promising candidate as a delivery vector for cancer therapy
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