IMPROVING EFFICACY OF GENE TRANSFER INTO HEMATOPOIETIC STEM CELLS

Hematopoietic stem cells (HSCs) are attractive targets for the gene therapy of a variety of genetic diseases. If successfully engrafted after ex vivo gene transfer and transplantation, they will generate a progeny of gene-corrected cells of all different blood lineages and potentially for the life span of the recipient. For gene therapy to be efficacious, effective gene transfer must be reached into HSCs without compromising their crucial biological properties. This is a challenge, because current transduction protocols require extensive ex vivo HSC culture and manipulation. HIV-1 derived vectors (lentiviral vectors, LV) may provide the means to achieve gene transfer in the majority of HSCs in a transplant, with minimal cell culture. In recent studies we have developed a protocol for efficient gene transfer into human cord blood-derived HSCs by LV, and we verified in a mouse xenotransplant model that our protocol did not affect the fundamental properties of HSCs. Our results provided a formal demonstration that primitive human HSCs with self-renewal and multi-lineage repopulation capacity can be efficiently gene-corrected by LV. However, we now need to reproduce our studies with HSC derived from more clinically relevant sources, such as the bone marrow and mobilized peripheral blood, and using more stringent xenotransplant models that allow longer follow-up time. We also found that, in hematopoietic progenitors, the proteasome, a cellular disposal, exerts an anti-viral activity, limiting the efficiency of LV gene transfer. We will investigate the mechanism by which proteasome limits LV gene transfer, in order to find a way to manipulate it and further increase gene transfer in HSCs. Gene therapy of some storage disorders and the hemoglobinopathies, however, may require very high levels of engraftment by gene-corrected HSC, a challenge even for an efficient gene transfer system. Thus, we will also explore strategies to amplify in vivo the ex vivo gene-modified HSCs. If successful, these studies will validate LV for highly efficient gene transfer into primitive HSCs, providing the scientific rationale for a clinical trial of this new gene transfer system.