Ex vivo expansion of hematopoietic stem cells for use in nonmyeloablative transplantation

Hematopoietic stem cell transplantation (HSCT) is used to treat a wide range of hematologic and non-hematologic disorders. Recently, interest has grown in the potential of autologous HSCT coupled to gene therapy for the treatment of genetic blood disorders as a way of avoiding the severe immunologic reactions associated with allogeneic HSCT. However, the remaining risks in using myeloablative conditioning regimens to allow relatively small numbers of transplanted HSCs to be transplanted greatly limit the applicability of this approach. Nonmyeloablative regimens would be an appealing alternative but necessitate the generation of large numbers of genetically corrected HSCs to achieve therapeutic levels of chimerism. In this thesis I have explored the potential of forced overexpression of homeobox genes as a strategy to obtain the degree of HSC expansion required. In a first series of experiments, I found that HOXB4 and NUPHOX transduced and expanded HSCs maintain the ability of fresh HSCs to produce sustained, high level, polyclonal, lympho-myeloid chimerism when transplanted into mice given 2-2.5 Gy. I then tested the therapeutic efficacy of ex vivo expanded HSCs in nonmyeloablated mice with severe beta-thalassemia caused by the homozygous deletion of the beta-major globin gene (beta-MDD). The results of these experiments showed that this approach could produce a dramatic improvement in the hematocrit, hemoglobin and RBC morphology and ultimately the cure of the thalassemic phenotype that was not achievable in recipients of equivalent numbers of unmanipulated BM cells or of cells transplanted immediately after transduction. Again, the cured mice displayed a sustained, high level of polyclonal chimerism. Together these data provide "proof of principle" of the curative potential of ex vivo expanded HSCs in a preclinical model of beta-thalassemia treated with nonmyeloablative conditioning.