Construction Of Recombinant NDV RClone30-CD And Investigation Of Its Tumor Inhibitory Ability

Cancer is the major disease that threaten human health, although the treatments for cancerhave been greatly improved and enhanced, which still could not significantly improve theefficiency of tumor suppression or improve the survival of patients. Traditional treatments areradiotherapy and chemotherapy, but they have a great risk, the course of treatment brings pain andside effects to patients. Newcastle disease virus (NDV) is a single strand non-segmented negativestrand RNA virus, belongs to the avian paramyxovirus genus, Paramyxoviridae andMononegavirals. In the1960s, the study found that NDV can be used for the treatment of humantumors, it replicates in tumor cells specifically, at the same time does not impact on normal cells.Reverse genetics technology has established and has a wide range of application. The researchersmodify the viral genome to increase the anti-tumor effect of wild-type strain. In this study, thesuicide gene CD inserted into the genome of the Newcastle disease virus, and the virus rescue byreverse genetics technique and coordinate with5-fluorocytosine (5-FC) to chieve the purpose ofsuppressing tumor.CD (Cytosine deaminase) is a member of the suicide gene family, it exists in E. coli or yeastgenome, which does not exist in mammals. The non-toxic prodrug5-FC can be converted to atoxic anticancer drug5-fluorouracil (5-FU) by CD,5-FU can inhibit tumor cells. CD gene wasisolated from E. coli JM109genome. Eukaryotic expression plasmid pEE12.4-CD-IRES-EGFP(pEE12.4IE-CD) was constructed. To verify biological activity of CD, HepG2cells transfectedwith pEE12.4IE-CD and treated with different concentrations of5-FC, MTT method was used todetect cell mortality in vitro, the results showed that CD has ability to transform5-FC and thegrowth of HepG2cells was inhibited significantly.Lentivirus and adenovirus are common carriers to deliver suicide genes into cells, both ofthem do not have anti-tumor ability. Newcastle disease virus was used in this study as a vector todeliver the CD gene into cells and enhance the anti-tumor ability. It is the first time to insert theopen reading frame of CD gene between the F gene and HN gene in the viral genome andconstructed a full-length cDNA clone of NDV with CD by reverse genetics technology. Thetranscript plasmid that carried the CD gene was named pBrClone30-CD. The transcription plasmidwas transfected with helper plasmids expressing nucleoprotein, phosphoprotein and large proteininto BHK-21cells which stably expressing T7RNA polymerase to rescue the NDV. The viruses proliferated in SPF chicken embryo, the allantoic fluid was collected for hemagglutination andhemagglutination inhibition assay, the recombinant Newcastle disease virus was named NDVrClone30-CD strain. The rescued viruses were passaged in embryo eggs for eight times, the viraltiters were stable and can proliferated stably. The results indicated that the insertion of exogenousgene CD did not affect the replication character of recombinant virus.In vitro experiment, the recombinant NDV rClone30-CD was tested for inhibiting HepG2cells by MTT assay at different MOI. The result showed that recombinant virus could inhibit thegrowth of HepG2cells in a dose-dependent manner. At high MOI, inhibiting effect ofrClone30-CD/5-FC was significantly greater than rClone30-CD, recombinant NDV exhibitedsignificant synergistic effect with prodrug5-FC.In vivo experiment, firstly, the maximum safe dose and median lethal dose (LD50) of5-FCwere measured to ensure the safety of the experimental mice and optimize the drug injection dose.The Kunming mice H22inguinal subcutaneous tumor models were established. The tumor-bearingmice were treated with the recombinant NDV rClone30-CD by intra-tumorous injection,5-FC byintra-tumorous or tail vein injection to achieve the purpose of co-treatment. Compared with thetreatment group of rClone30-CD, rClone30-CD/5-FC inhibited growth of tumor significantly,recombinant NDV rClone30-CD with prodrug5-FC synergistic effect is significant and also couldincrease the survival rate of mice. The results of different administrating methods were consistent,indicated that intra-tumorous injection or intravenous injection of5-FC were able to achieve thepurpose of inhibiting tumor.5-FU in blood samples of mice were determined, the presence of5-FUwas detected in rClone30-CD/5-FC group, but not found in5-FC group. The results showed thatthe CD gene inserted into the NDV converted5-FC to5-FU, and the anti-tumor effect ofrClone30-CD/5-FC increased significantly due to the presence of5-FU.In the present study, we successfully rescued the recombinant NDV rClone30-CD strain byreverse genetics technology. It was confirmed that the recombinant NDV rClone30-CD/5-FC hadability to inhibit HepG2cells effectively in vitro. In vivo study, two different methods ofadministration of5-FC were used, rClone30-CD/5-FC was able to inhibit tumor cells and extendedthe survival time of experimental mice. Of this study was to increase the anti-tumor effect of theNDV lentogenic strain. This study established the platform for using recombinant NDV as a vectorfor suicide genes to utilize in the targeted treatment of cancers.