Delivering nucleases in the form of mRNA is particularly attractive because it ensures that the potentially toxic nucleases are present only transiently. Moving beyond the issue of gene delivery, our study demonstrated proof of concept for the process of ex vivo gene therapy, wherein a patient’s cells are isolated, genetically modified in vitro and then reintroduced into the patient. Admittedly, challenges must be overcome before translation to the clinic. A feature critical to the success of ex vivo gene therapy is availability of a culturing method, something that had been established for rodent SSCs, but that is an area of fervent yet controversial investigation for human SSCs. Regarding correction of genes other than GFP, other examples of genome engineering at a model target locus have generally proven to be highly relevant in predicting applicability to new loci. In the case of genetic defects causing spermatogenic failure, corrected SSCs would have a selective advantage and may not need to be enriched before transplantation as only corrected SSCs would produce sperm. For diseases in which corrected cells do not have a selective advantage, strategies described above could facilitate enrichment of rare cells with the corrected loci. Many genetic diseases affect cells or tissues for which a cognate stem cell type is unknown and or impossible to culture. Many diseases also are systemic in nature, affecting numerous cell types that cannot be treated with a single cell type. Furthermore, even for certain diseases of the blood that have been successfully treated by genetic modification and transplantation of autologous hematopoietic stem cells, patients then live with the burden of potentially passing the heritable trait on to their children. Ultimately the most permanent and all-encompassing cure of a genetic disease would be to genetically modify the germ cells. The use of SSCs in our study is especially unique because unlike all other adult stem cell types, SSCs are capable of generating sperm, which carry genetic information to the next generation. SSCs are also unique in their developmental plasticity, capable of transdifferentiation to multiple lineages or even to pluripotency without the use of exogenous genetic factors. An obvious application one could imagine is treatment of infertility caused by mutations affecting germ cell development. However, several substantial issues would need to be addressed before applying germline gene therapy in humans. First, our transplantation study revealed that the gene-corrected cells appeared to R428 1037624-75-1 exhibit attenuated differentiation capability. The cause of this shortcoming is unclear. Previous studies showed that GS cells can be transfected and passaged extensively in vitro while still retaining the ability to produce spermatozoa following transplantation. Our data did not distinguish among potential effects of technical issues with the transplant recipients, genetic or epigenetic effects on the cells unrelated to genome editing, or unintended genomic changes related to off-target cutting by the ZFNs. In other experiments we have found that delivering ZFNs via DNA expression vectors causes gross chromosomal rearrangements.