453. Reproductive Safety Evaluation and Quantification of Adenoviral Vectors in the Mouse

Expression of transgene other than in the target tissue may cause side effect and safety problems in gene therapy. This is particularly important if vector distributes to gonads, raising the possibility of inadvertent germ-line transmission. In addition, for indications such as prostate cancer and ovarian cancer, the proximity of the point of viral administration to organs of the reproductive system raises concerns regarding inadvertent germ-line transmission of genes carried by the virus. To evaluate the reproductive toxicity of in vivo adenovirus mediated gene transfer, we studied the biodistribution and potential germ-line transmission of p53-expressing adenovirus (Ad-CMV-p53). Both male and female Balb/c mice were injected with 1x 10 9 PFU of Ad-CMV-LacZ or Ad-CMV-p53. DNA and RNA extracted from major organs including gonadal tissues were analyzed for vector sequences and expression. The PCR analysis showed that there were detectable vector sequences in liver, kidney, spleen, seminal vesicle, epididymis, prostate, ovary, and uterus. The RT-PCR analysis showed that Ad-CMV-LacZ or Ad-CMV-p53 viral RNA were present in spleen, prostate and ovary. Direct injected male and female mice of adenovirus vector into testis and ovary were mated and their offspring were evaluated for germ-line transmission of the adenoviral vector. The PCR analysis showed no evidence of germ-line transmission, although vector sequences were detected in DNA extracted from gonadal tissues. Quantitative PCR was used to determine adenovirus level over period of several weeks. Real-time PCR result confirmed a significant decrease of adenovirus in all tissues tested 1 week after injection. We have also developed for the cell specific localization of viral DNA in gonad tissues by using in-situ PCR. No positive signals were detected in germ cell related cells inside seminiferous tubules and follicles. These data provide strong evidence that the risk of the inadvertent germ-line transmission of vector sequences following intraperitoneal or direct injection into genito-urinary system of adenovirus is extremely low, although vector distributed to gonadal tissues.