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Adeno-associated virus (AAV)-mediated transduction of male germ line stem cells results in transgene transmission after germ cell transplantation

Ali Honaramooz^*,1,2, Susan Megee^*,1, Wenxian Zeng^*, Margret M. Destrempes, Susan A. Overton, Jinping Luo^*, Hannah Galantino-Homer^*, Mark Modelski^*, Fangping Chen^*, Stephen Blash, David T. Melican, William G. Gavin, Sandra Ayres, Fang Yang, P. Jeremy Wang, Yann Echelard and Ina Dobrinski^*,3

^* Center for Animal Transgenesis and Germ Cell Research, Department of Clinical Sciences, New Bolton Center, and

Department of Animal Biology, School of Veterinary Medicine, University of Pennsylvania, Kennett Square, Pennsylvania, USA;

GTC Biotherapeutics, Framingham, Massachusetts; and

Tufts Cummings School of Veterinary Medicine, North Grafton, Massachusetts, USA

³ Correspondence: Center for Animal Transgenesis and Germ Cell Research, University of Pennsylvania, 382 W. State Road, Kennett Square, PA 19348, USA. E-mail: dobrinsk{at}vet.upenn.edu

We explored whether exposure of mammalian germ line stem cells to adeno-associated virus (AAV), a gene therapy vector, would lead to stable transduction and transgene transmission. Mouse germ cells harvested from experimentally induced cryptorchid donor testes were exposed in vitro to AAV vectors carrying a GFP transgene and transplanted to germ cell-depleted syngeneic recipient testes, resulting in colonization of the recipient testes by transgenic donor cells. Mating of recipient males to wild-type females yielded 10% transgenic offspring. To broaden the approach to nonrodent species, AAV-transduced germ cells from goats were transplanted to recipient males in which endogenous germ cells had been depleted by fractionated testicular irradiation. Transgenic germ cells colonized recipient testes and produced transgenic sperm. When semen was used for in vitro fertilization (IVF), 10% of embryos were transgenic. Here, we report for the first time that AAV-mediated transduction of mammalian germ cells leads to transmission of the transgene through the male germ line. Equally important, this is also the first report of transgenesis via germ cell transplantation in a nonrodent species, a promising approach to generate transgenic large animal models for biomedical research—Honaramooz, A., Megee, S., Zeng, W., Destrempes, M.M., Overton, S.A., Luo, J., Galantino-Homer, H., Modelski, M., Chen, F., Blash, S., Melican, D. T., Gavin, W. G., Ayres, S., Yang, F., Wang, P. J., Echelard, Y., Dobrinski, I. Adeno-associated virus (AAV) -mediated transduction of male germ line stem cells results in transgene transmission after germ cell transplantation. FASEB J. 22, 374–382 (2008)

Key Words: transgenesis • testis • mouse • goat