Assessment Of Efficiency And Safety Of A Novel Oncolytic Adenovirus M1 In Vivo And Study On Targeted Delivery Of Adenoviral Vector

PartⅠCharacterization of the kinetics of viral distribution and spread after different injection of adenovirus Adv-GFP in a BALB/c mice model[Objective] To explore the kinetics of viral distribution and spread in a BALB/c mice model after the intravenous infusion or retroperitoneal infusion, and evaluate the safety of reconstructed adenovirus administration with liver transplantation in hepatocellular carcinoma therapies.[Methods] BALB/c mice were intravenously or retroperitoneally injected with purified Adv-GFP in order to make animal models. The expression of green fluorescence protein (GFP) and the adenovirus mRNA was determined using fluorescence assay and in situ hybridization. The virus in serum and liver was tittered at the indicated time point.[Results] The expression of GFP and adenovirus mRNA were both located preferentially in liver, spleen, lung and kidney by two methods of infusion in mice. There were more adenovirus vectors in liver after the retroperitoneal infusion than that after intravenous infusion. Though there was virus dissemination into the general circulation at early time points after retroperitoneal infusion, the amount of virus in liver was higher in 1w than that after intravenous infusion.[Conclusion] The retroperitoneal infusion of adenovirus vectors may be achieve better treatment and reduce systemic reaction in a certain extent, which is more suitable for gene therapy.PartⅡBiodistribution and kinetics of novel selective oncolytic adenovirus M1 via systemic administrationOncolytic adenoviruses represent a promising novel therapeutic option for the treatment of cancer. Despite rapid translation into human clinical trials and demonstrated safety, the fundamental properties of oncolytic adenovirus biodistribution, spread, viral persistence and replication in vivo have not been completely elucidated. The aim of this study was to evaluate the kinetics of viral distribution, spread, replication and anti-tumoral efficacy as a single agent after intravenous administration of a novel oncolytic mutant M1, the E1A CR2-deleted Adv5 with a fragment of antisense plk1 cDNA inserted into the deleted 6.7K/gp19K region, which combine oncolytic properties with efficient plk1 silencing in our previous reports. In the present study, we established a new human orthotopic gastric carcinoma with high frequency metastasis mouse model and demonstrated that M1 spread not only in the local primary tumor but also in the disseminated metastasis, on the other hand, M1 could effectively replicate in tumor cells leading to"oncolysis"and eliminate the targeted gene plk1 expression in human orthotopic gastric carcinoma model mice and therefore, if intravenous administration at early time points, could prolong the survival time of tumor-bearing mice.PartⅢEffects of conditionally replicating adenovirus on isolated human umbilical endothelial cell[Objective] This study was to explore the effects of conditionally replicating adenovirus on proliferating and nonproliferating endothelial cells.[Methods] HUVECs were isolated by filling umbilical veins with digestive enzyme solution. HUVECs were cultured and observed. Endothelial cells were identified by immunofluorescence staining and scanning electron microscope examination. The oncolytic effects of Adv5/dE1A-Aschk1 on endothelial were measured by cytopathic effect assay (CPE) and were confirmed by quantitave cytotoxicity assay using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide method. Cell apoptosis was measured by flow cytometric assay (FCM).[Results] High-purified endothelial cells were isolated. Adv5/dE1A-Aschk1 lysed Hela cells in a replication-dependent fashion, but did not exhibit detectable cytopathic effects on nonproliferating endothelial cells (Q-HUVEC) at a MOI of 500. The growth inhibitory effects on endothelial cells increased with the increase of viral titer. The growth inhibition rate of P-HUVEC is 22±2.5% with Adv5/dE1A-Aschk1 at a MOI of 100 for 96 hours, but that of Q-HUVEC is 13±2.3%.The combination of Adv5/dE1A-Aschk1 and irradiation resulted in cell apoptosis of Q-HUVEC (23.1±2.5%) and P-HUVEC (35.7±3%).[Conclusions] HUVECs may be taken as a good target for the research of conditionally replicating adenovirus. The viral tier of injuring endothelial cells is much higher than that of killing tumor cells, so Adv5/dE1A-Aschk1 has a wide range of therapeutic dosage. And it preferentially replicates in and lyse P-HUVEC compared to Q-HUVEC.PartⅣAssessment of efficiency and safety of a novel oncolytic adenovirus M1 in vivo[Objectives] To evaluate the safety of a novel oncolytic mutant M1 after intravenous and retroperitoneal administration, and to investigate the efficiency of reconstructed adenovirus in immunosuppressive therapies.[Methods] Animal models were established by intravenously and retroperitoneally injected with purified M1. At different time points, Blood samples were taken from the mice to test the liver and renal function; Microscopic examination of liver was performed to observe the pathological changes. Immunohistochemical method was used to examine the expression of adenovirus in liver. Lymphocytes infiltration in liver and activation of adenovirus specific T cells were also examined.[Results] No signs of general toxicity were seen following injection of M1, with a transient increase of ALT and Cr. Microscopic examination revealed mild inflammatory response in liver. There were more adenovirus vectors in liver after the retroperitoneal infusion than that after intravenous infusion. Cyclosporine A significantly lowers the increase of ALT and Cr resulted from adenovirus, and effectively upgrades adenovirus in mouse liver concentration.[Conclusions] The novel oncolytic mutant M1 is safe in vivo, and the use combined with immunosuppressive therapy could enhance its effectiveness and safety.PartⅤStudy on targeted delivery of adenoviral vector[Objective] To explore a new way to improve viral uptake rates in tumor antigen-specific CTL cells for a novel oncolytic adenovirus M1.[Methods] We fused Tat to the ectodomain of the coxsackievirus-adenovirus receptor (CARex) to attach Tat to adenoviral fiber knobs. Purified fusion protein was used to facilitate gene transfer in tumor antigen-specific CTL cells.[Results] Tumor antigen-specific CTL cells were isolated and induced. pcDNA-CARex-Tat was constructed and the fused protein was purified. CARex-Tat improved viral uptake rates in permissive cell HeLa(P<0.05), but not significantly enhance transfection efficiency of NIH-3T3(P>0.05); CARex-Tat can enhance transfection efficiency of K562 cells, but almost not improve the low transfection efficiency in tumor antigen-specific CTL cells.[Conclusions] Isolation and induction of tumor antigen-specific CTL cells are practicable, but the valuable tool to overcome natural tropism of vectors needs to be further explored.