Top of pageAbstract The recent observation of vector sequences in the semen of men undergoing clinical gene therapy for hemophilia has highlighted the need to thoroughly evaluate the risk of inadvertent germline transduction in a clinically-relevant animal model. While previous studies have demonstrated that adenoviral and AAV vectors do not modify mature sperm cells, studies are lacking which examine the effects of murine retroviral vectors on mature sperm cells and also the immature male germ cells that ultimately give rise to sperm. We have used three different approaches to investigate whether the nascent fetal germline may be at risk of inadvertent alteration following a direct vector injection approach to in utero gene therapy in the sheep model. First, we conducted breeding studies and analyzed the tissues from the 10 resultant offspring for the presence of vector sequences. All organs tested were devoid of proviral DNA. While these results suggested that the germline had not been altered, only 1-2 animals are obtained from each breeding and each offspring is produced from a single germ cell from each parent, making it difficult to use this approach to obtain a definitive answer regarding germline alteration. For this reason, the second approach we used was to perform PCR analysis on OviPure™ gradient-purified sperm cells from these sheep. While in breeding experiments each lamb is derived from a single sperm cell, each semen collection contains roughly 160×10^6 sperm cells. Thus far we have analyzed the ejaculates from 19 in utero transduced rams. The purified sperm cell pellet from six of these sheep was PCR positive for proviral DNA, again suggesting the fetal germline had been modified. However, since this purified population can still contain up to 0.1% hematopoietic cell contamination, we performed RT-PCR for CD45 on RNA isolated from the purified sperm cells to assess hematopoietic contamination. While the purified sperm cell populations from 2 of the sheep were contaminated with significant levels of hematopoietic cells, the remaining 4 were not, suggesting that the vector sequences were within the actual sperm cells. To further purify the sperm cells, we performed a forensic DNA preparation on the OviPure™ purified sperm cells to remove any residual somatic cells. The forensically purified sperm cell pellets from the same four sheep were still PCR positive for vector sequences, providing compelling evidence that the sperm cells themselves had been transduced. We are currently using cell sorting to purify sperm cells from these 4 sheep for PCR analysis. As a third approach, we performed immunohistochemistry (IH) on sections of the testis collected from in utero transduced sheep. Numerous somatic cells within the male reproductive tissues were transduced. We have also observed very low levels of germ cells that are transgene positive. In conclusion, our analysis on over 3×10^9 sperm cells suggests that the direct injection approach employed in these studies may result in the transduction of very low numbers of male germ cells. It is hoped that these ongoing studies will provide a clear picture of what the risks of fetal germline modification may be following in utero gene transfer.