757. Stable In Vivo Transduction of Primitive Hematopoietic Stem Cells After Mobilization and Intravenous Injection of an Integrating Gene Transfer Vector

Current protocols for hematopoietic stem cell (HSC) gene therapy involve the collection of HSCs from donors/patients, in vitro culture, transduction with retrovirus vectors, and retransplantation into myelo-conditioned patients. Besides its technical complexity, disadvantages of this approach include the necessity for culture in the presence of multiple cytokines which can affect the pluripotency of HSCs and their engraftment potential. Furthermore, the requirement for myeloablative regimens in patients with non-malignant disorders creates additional risks.We therefore explored the potential for in vivo transduction of HSCs. We developed an approach that involves GCSF/AMD3100-mediated mobilization of HSCs from the bone marrow into the peripheral blood stream, followed by intravenous injection of a Sleeping Beauty transposase-based integrating helper-dependent adenovirus (HD-Ad5/35++) vector system. These vectors target CD46, a receptor that is expressed at higher levels on HSCs than on more differentiated bone marrow and blood cells. We demonstrated in human CD46 transgenic mice and immunodeficient mice with engrafted human CD34+ cells that HSCs transduced in the periphery home back to the bone marrow where they persist and stably express the transgene long-term. In the CD46 transgenic mouse model we showed that our in vivo HSC transduction approach allows for the stable transduction of primitive HSCs, i.e. cells capable of forming multi-lineage progenitor colonies. At 12 weeks after in vivo transduction, we detected GFP marking in bone marrow HSCs in the range of 1 to 2%. Importantly, the proportion of transduced primitive HSCs increased over time. Furthermore, in vivo transduced HSCs were able to repopulate the hematopoietic system of lethally irradiated C57BL/6 mice, showing the functionality of the modified HSCs. Our in vivo HSC transduction approach did not result in innate toxicity or significant transduction of non-hematopoietic tissues. Genome-wide integration site analysis in in vivo transduced HSCs revealed a close-to-random integration pattern without preference for genes and the absence of integration into or near to cancer-associated genes.In conclusion, our novel in vivo transduction approach allows for stable genetic modification of primitive HSCs without the need of ex vivo culture, myelo-conditioning, and transplantation. Thus, our method is relevant for a broader clinical application of gene therapy of inherited diseases as well as infectious diseases and cancer.