This application addresses the broad Challenge area (15): """"""""Translational Science"""""""" and the specific Challenge Topic (15-DK-102): """"""""Develop Improved Animal Models of NIDDK Diseases"""""""". The lack of large animal models of genetic diseases is a serious barrier to understanding the underlying pathophysiology necessary for devising innovative therapies, and the reliable testing of the safety and efficacy of these therapies. While recapitulating some phenotypic aspects of human disease, mouse models have not accurately reflected the spectrum of disease in conditions such as Fanconi anemia. There are currently no large animal systems whose genomes can be readily manipulated. This work will generate a """"""""toolbox"""""""" of protocols, cell lines and reagents to bridge this void, and will be made available to NIH researchers to facilitate the future development of large animal models of genetic diseases. Currently the largest barrier to developing large animal models that faithfully recapitulate human disease is the derivation of viable animals from modified stem cells. While generating large animals from embryonic stem cells or by somatic cell nuclear transplant has been fraught with difficulties, we have demonstrated transplanting spermatogonial stem cells (SSC) results in long-term production of donor derived sperm. Thus, we will take several parallel paths to exploit this approach to derive the tools to generate genetically modified dogs, focusing on the future generation of a Fanconi anemia dog as a test of principle. To accomplish this goal, we will utilize key technologies, including Adeno-associated virus (AAV) mediated homologous recombination (HR) and the in vitro expansion of SSCs, prior to transplantation. Thus, conditions to culture SSC without losing their potential to generate viable sperm will be developed and then assayed in vivo. Using these conditions we will develop virally """"""""tagged"""""""" SSC lines that allow the tracking of SSC, or of their progeny. These will be powerful tools for any investigator interested in canine development or animal models. To assess the role of disrupting FANCB in canine cells we will use AAV mediated HR. AAV is increasingly recognized as an efficient tool for generating transgenic animals and knockout animals both in vitro, and upon direct injection in vivo. AAV exonic gene trap vectors lead to efficient modification of targeted genes. This technology will be used to knock out function of the X-linked FANCB gene, leading to a model for Fanconi anemia in dog cells. Once generated, the molecular and biochemical phenotype of these cells needs to be analyzed. Concurrently, reagents and assays will be developed for defining the Fanconi anemia phenotype in canine cells. Vectors optimized in vitro will be used to infect and enrich for SSC in preparation for transplantation and generation of genetically modified dogs. Finally, though we demonstrated transfer of SSCs from a mixed population of cells, this has not been accomplished with purified SSCs that would result from genetically modified cells after in vitro expansion. Thus we will address several hypotheses regarding the relationship of cell dose and the resultant effect on contribution to donor derived sperm, and whether non-germ cells play a role in SSC engraftment and expansion post transplantation. With each Aim progressing independently, we will be able to develop protocols to generate improved animal models of human disease within the timeframe of this award. Similarly, pursuing parallel in vitro and in vivo protocols for generating genetically modified dogs will expedite progress. Finally, as stimulating the economy is an objective of this RFA, we will create, and help retain jobs, focus on purchasing United States manufacturers and support core services at the Fred Hutchinson Cancer Research Center and the Cornell University. Mice have long provided a model for human genetic diseases because the technology exists to manipulate the mouse genome. However, mouse physiology is not identical to that of humans, and in many cases, replicating a human genetic defect in a mouse does not replicate the associated disease state. The dog has provided a more reliable model of human physiology in pre-clinical research. However, genetic manipulation of dog or other large animal models has been unsuccessful thus far. This proposal aims to combine the advantages of both models with the new technology of spermatogonial stem cell (SSC) transplantation. SSC are the cells residing in the testis that continue to generate sperm for the life of the animal. In mice, SSC can be cultured from a donor testis, genetically altered, and transplanted into the testis of a host. Mating of the host mouse produces transgenic offspring, carrying the altered genetic information. We plan to optimize the methods for culture, genetic manipulation, and transplantation of SSC in dogs, and to use this technology to set the stage for producing a canine model of Fanconi Anemia.
Mice have long provided a model for human genetic diseases because the technology exists to manipulate the mouse genome. However, mouse physiology is not identical to that of humans, and in many cases, replicating a human genetic defect in a mouse does not replicate the associated disease state. The dog has provided a more reliable model of human physiology in pre-clinical research. However, genetic manipulation of dog or other large animal models has been unsuccessful thus far. This proposal aims to combine the advantages of both models with the new technology of spermatogonial stem cell (SSC) transplantation. SSC are the cells residing in the testis that continue to generate sperm for the life of the animal. In mice, SSC can be cultured from a donor testis, genetically altered, and transplanted into the testis of a host. Mating of the host mouse produces transgenic offspring, carrying the altered genetic information. We plan to optimize the methods for culture, genetic manipulation, and transplantation of SSC in dogs, and to use this technology to set the stage for producing a canine model of Fanconi Anemia.
| Harkey, Michael A; Asano, Atsushi; Zoulas, Mary Ellen et al. (2013) Isolation, genetic manipulation, and transplantation of canine spermatogonial stem cells: progress toward transgenesis through the male germ-line. Reproduction 146:75-90 |
| Fortier, Lisa A; Travis, Alexander J (2011) Stem cells in veterinary medicine. Stem Cell Res Ther 2:9 |
