Biodistribution And Safety Assessment Of Bladder Cancer Specific Recombinant Oncolytic Adenovirus In Nude Mice

Background:Bladder cancer is one of the most common tumors in urinary systerm, it’s mobidity is yearly rising and the recurrence rate after treating is comparably high. Gene therapy based on bladder-specific conditionally replication competent adenoviruses are attractive therapeutics for bladder cancer. For oncolytic adenoviruses, besides the specific efficacy, the toxicity and safety are equally important. The previous works about safety evaluation for constructed bladder tissue specific adenovirus are poorly documented. Collection of data from preclinical studies to access the spread of adenovirus E1A transgene expression outside tumor tissue is of the utmost importance in assessing the risks associated with gene therapy. Thus, we investigated the amplification, purification, identification, biodistribution and body toxicity of bladder specific oncolytic adenovirus Ad-PSCAE-UPⅡ-E1A (APU-E1A) and Ad-PSCAE-UPⅡ-E1A-AR (APU-E1A-AR), providing meaningful information prior to embarking on human clinical trials.Materials and Method:Conditionally replicate recombinant adenoviruses (CRADs) APU-E1A, APU-EIA-AR were constructed with bladder tissue specific Uroplakin Ⅱ (UP Ⅱ) promoter to induce the expression of Ad5E1A gene and E1A-AR fusing gene, and PSCAE was inserted at upstream of promoter to enhance the function of promoter. To examine adenovirus replication and distribution in mice, we also constructed the detection adenovirus Ad-PSCAE-UPⅡ-LUC (APU-LUC). These adenoviruses were propagated in HEK293cells and purified by cesium chloride gradient centrifugation. E1A, UP Ⅱ, PSCAE, PSCAE, AR, UPⅡ-El A and UP Ⅱ-LUC gene fragments in adenoviruses were indentified with PCR, and the viral titer was determined by TCID50(50%Tissue Culture Infective Dose) method. Based on the cytopathic and anti-tumor effect of bladder cancer, subcutaneous xenografts tumor in nude mice were made by human bladder cancer cell line EJ injection, then different doses (5×107pfu,5×108pfu,5×109pfu) of oncolytic adenoviruses (APU-E1A, APU-E1A-AR and APU-LUC) were intratumorally injected into xenografts. We then determined the toxicity through animal’s body weight, general health and behavioral assessment, hematological (percentage of lymphocytes in white blood cell and platelets) and hepatic (Aspartate aminotransferase, AST and Alanine aminotransferase, ALT) toxicity evaluation, organ coefficient, macroscopic and microscopic postmortem analyses (H&E and immunohistochemical staining). The spread of the transgene E1A of adenoviruses were detected with qRT-PCR and Western blot. Virus replication and distribution were examined with APU-LUC administration and Luciferase Assay.Results:After HEK293were transfected with oncolytic adenoviruses APU-E1A and APU-E1A-AR, the HEK293cells showed significant cytopathic effect (CPE), which confirmed the propagated reaction. After the adenoviruses were purified, OD260/OD280of viruses were1.2-1.24, TCID50/VP were0.98%-1%, and the higest infection unit was1.2×1012IU/ml. The expressions of gene E1A, UP Ⅱ, PSCAE, AR, UP Ⅱ-E1A and UP Ⅱ-LUC fragment were stable. Comparing with the control group, animal’s body weight of groups which were injected with APU-E1A and APU-E1A-AR were rising stably, and general assessment did not reveal any alteration in general behavior. The hematological alterations (percentage of lymphocytes in white blood cell and platelets) of groups which were injected with5×107、5×10pfu or higher dose (5×109pfu) of APU-E1A and APU-E1A-AR showed no difference in comparison with PBS group, and only slight increased transaminases (AST and ALT) in contrast to PBS group at5×109pfu of APU-E1A and APU-E1A-AR were observed. After adenoviruses injection, through dissection and general observations, the major organs of animals such as heart, liver, spleen, lung, kidney, brain, stomach, testicles, seminal vesicle, thyroid, bladder and urethra were not observed any abnormal phenomena such as hemorrhage, necrosis and swelling. Statistics of average organ coefficient of animals had no significant difference compared with control group. H&E staining and immunohistochemical staining for adenovirus E1A gene showed that the oncolytic adenoviruses APU-E1A, APU-E1A-AR did not replicate and express outside of xenograft tumor tissue. E1A transgene were detected highly expressed in tumor and slightly expressed in liver tissue, and E1A protein did not disseminate to organs outside of xenograft tumor. Expression of LUC was much higher in xenograft tumor tissue comparing with other organs, which had significant statistical difference.Conclusions:Our study showed that the recombinant oncolytic adenoviruses APU-E1A-AR and APU-E1A could be amplified in HEK293cells, the purity and titer of adenoviruses could meet the demand. The expressions of gene fragment E1A, UPⅡ, PSCAE, AR, UPⅡ-E1A and UP Ⅱ-LUC were stable after amplification and purification. APU-E1A-AR and APU-EIA had bladder cancer tissue specificity and appeared safe with5×107pfu and5×108 pfu intratumorally injection in mice, without any discernable effects on general health and behavior, hematological (percentage of lymphocytes in white blood cell and platelets) and hepatic (AST and ALT) evaluation, organ coefficient, macroscopic and microscopic postmortem analyses, as well as the E1A transgene and protein expression.