826. Non-Viral Gene Therapy Targeting Fanconi Anemia Hematopoietic Stem Cells: A Realistic Hope?
Authors: Meenakshi Noll, Ryan Bennett, Stephen Yant, Jacob G. Mikkelsen, Cindy Chen, Mark A. Kay, Markus Grompe
Journal: Molecular Therapy (2004)

The life span of patients with Fanconi Anemia (FA) is severely shortened by bone marrow failure and malignancies. Currently, the only efficient therapy is bone-marrow transplantation from a matched sibling donor. Therefore, FA is considered a good candidate disease for hematopoietic gene therapy. Our previous preclinical gene therapy experiments with Fancc knock-out mice have shown that wild-type hematopoietic stem cells (introduced by transplantation or generated by gene transfer) have a strong selective growth advantage in vivo. However, despite the successful modeling of FA gene therapy in the mouse, application of the same approach to humans is complicated by the requirement for ex vivo manipulation and scarcity of HSCs in FA patients. Consequently, the development of new gene transfer methods that support stable integration of therapeutic transgenes in vivo are greatly desired. Here, we have utilized the Sleeping Beauty (SB) transposon system to facilitate stable, transposase-mediated gene transfer of the human FANCC cDNA in vivo from a plasmid-based system. The transposase and FANCC transposon plasmid constructs were co-transfected into bone-marrow cells of Fancc mutant mice by two different methods: 1) via electroporation into enriched hematopoietic cells; 2) via direct injection into the femur cavity. In each case, the presence of the transgene was followed in treated animals by semi-quantitative PCR. Although no transgene was detectable in peripheral blood DNA 2 weeks after injection, 2/4 mice injected into the femur cavity were PCR positive 4 weeks after injection, even in the absence of selective pressure. At this time, a single low dose of cyclophosphamide (CPA) was given to provide a selective advantage for FANCC corrected cells. Four weeks after CPA administration, all the experimental electroporated animals (N=2) and femur injected animals (N=8) were strongly PCR positive in their peripheral blood and have remained stably positive for at least 9 months. Furthermore, upon transplantation of bone marrow from these mice into wild-type lethally irradiated recipients, we observed that 81/110 (74%) of day 12 CFU-S colonies were transgene positive. Similarly, 64/110 (64%) of the 12-day CFU-S colonies were transgene positive from the femur-injected donors, thus confirming that transgene integration had occurred in bona fide stem cells. Collectively, these studies demonstrate that a) hematopoietic stem cells from Fancc mutant mice are proficient in taking up plasmid DNA; b) stable integration of plasmid DNA into the genome of hematopoietic stem cells is possible and c) the efficiency of stable gene transfer using these nonviral approaches compares favorably to that achieved by alternative viral-based methods. We are in the process of analyzing transposon host cellular DNA junctions to determine the mechanism of transgene persistence in each experimental group. The above experiments have been repeated independently with more animals: N=5 for electroporated and N=6 for femur injected. Details of all these results will be discussed.