Identification of Optimal Insertion Site in Recombinant Newcastle Disease Virus (rNDV) Vector Expressing Foreign Gene to Enhance Its Anti-Tumor Effect

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.

Rapid Generation of Multiple Loci-Engineered Marker-free Poxvirus and Characterization of a Clinical-Grade Oncolytic Vaccinia Virus

“…Finally, marker genes and other inserted sequences in viral vectors may affect the overall fitness of the resulting virus, as shown in a number of cases. 47 , 48 , 49 , 50 , 51 , 52 Therefore, our study has confirmed the great advantages of the SEM system for the removal of marker genes.…”

Oncolytic Viruses—Interaction of Virus and Tumor Cells in the Battle to Eliminate Cancer

“…Although NDV is naturally tumor-selective, there are inherent problems with the virus which need to be overcome in order to improve its ability to infect and spread within solid tumors as viral spread is limited by factors such as the extra-cellular matrix ( 95 ). Again, there are many studies looking at ways to modify this virus to make it safer and more efficacious; for example, insertion of the IL-2 gene into the NP/P site has proved effective ( 96 ). Inclusion of IL-2 and/or TRAIL has been shown to increase apoptosis levels in infected cells resulting in the tumor-selective parental virus becoming an even more potent anti-cancer agent ( 97 – 101 ).…”

Armed oncolytic viruses: A kick-start for anti-tumor immunity

“…AdV [108] Neuroblastoma [45], Glioma [43], Prostate [48], Squamous Cell Carcinoma [46,47], Melanoma [108] Infiltration of macrophages, T helper, CTL and NK cells [43,45,47,48] No signs [44,46,47] HSV [30,[43][44][45]47,48] Improved survival [108] and protective against rechallenge [47] VSV [46] IL-2 NDV [52][53][54][55][56] Melanoma [54,55], Hepatoma [52,53], Squamous Cell Carcinoma [51] Infiltration of T helper and CTL [51][52][53][54][55][56] No signs [53,54] HSV [51] Immunity against rechallenge with tumor cells [51][52][53][54]…”

“…Systemic treatment with IL-2 is associated with major adverse side effects in humans [24]. Therefore, local delivery of IL-2 by OVs has been tested by several research groups [51][52][53][54][55][56]. In these murine studies, reduced tumor growth and increased T cell infiltration of the tumors was reported.…”

“…In addition to oncolytic activity, the efficacy of the OV immune therapy is influenced by the efficiency of a virus to express the immune modulators. For instance, for NDV it has been shown that the insertion site in the virus determines the expression levels of the transgene [56]. The effect of the insertion site may also hold true for other OVs, but is often not discussed.…”