Identification Of The Optimal Insertion Site In Recombinant Newcastle Disease Virus(rNDV) Vector Expressing Foreign Gene To Enhance Its Anti-tumor Effect

Recombinant Newcastle disease virus(rNDV)has been developed as a vector expressing foreign gene to enhance its anti-tumor efficacy.Previous studies found that the expression level of foreign gene may be influenced by insertion site in rNDV.However,the optimal insertion site for foreign gene expression to increase oncolytic effects remains unclear.In this present study,an infectious clone of the NDV(rClone30)was generated as a vector.Foreign genes including enhanced green fluorescence protein(EGFP)and Interleukin-2(IL2)were inserted into four different intergenic regions of rClone30 resuting in recombinant viruses(rClone30-EGFP and rClone30-IL2),respectively.The recombinant viruses were co-transfected with BHK21 cells together with helper plasmids and successfully rescured.The recoverd viruses were passaged ten times in embryonated SPF chicken eggs.The results showed that the viruses could stably replicate in SPF eggs and the insertion of foreign genes did not affect the growth characteristics of the recombinant virus.Then the expression levels and transcriptional levels of foreign genes in the HepG2,U251,A549 and He La cells infected with rClone30-EGFP and rClone30-IL2 at MOI of 10 were detected by fluorescence microscopy and Real-time PCR to identify optimal foreign gene insertion site,respectively.To further investigate the anti-tumor effects of rClone30 in vivo,the H22 hepatic carcinoma solid tumor model was established.The tumor-bearing mice were intratumorally injected with the five recombinant viruses named as rClone30,rClone30-IL2-NP/P,rClone30-IL2-P/M,rClone30-IL2-M/F and rClone30-IL2-F/HN,and the mice tumnor volume and survival rate were determined.At the same time,the percentage of CD4~+T and CD8~+T isolated form mock-treated and virus-treated mice spleen was detected using flow cytometry.The results showed that rClone30-EGFP and rClone30-IL2 were successfully rescued.There wasn't significant difference between rClone30s and parental virus in growth kinetics.The mRNA transcriptional and protein expression levels of foreign genes were most abundant in HepG2 cells,when they were inserted between the NP and P genes of the rClone30s.In order to further evaluate its optimal insertion site to deliver IL2 for cancer therapy,the H22-oxter-tumor-bearing C57BL/6J mice were treated with rClone30-IL2s to examine the oncolytic effects when IL2 was inserted in different gene junctions.The results showed that rClone30-IL2-NP/P was most effective in inhibition of murine hepatoma carcinoma tumors,with the inhibition rate arriving at 98.25%.In addition,the mice treated with rClone30-IL2-NP/P exhibited the highest percentage of CD4~+T(10.63%)and CD8~+T cells(19.36%)among the rClone30-IL2-treated groups,revealing that NP and P gene junction may be the optimal insertion site for foreign gene expression to elevate the T cells immune response.To sum up,these results demonstrate that NP and P gene junction in rClone30 is the optimal insertion site for foreign genes expression in inhibition of tumor.