Therapeutic hemoglobin synthesis in beta-thalassemic mice expressing lentivirus-encoded human beta-globin

The stable introduction of a functional globin gene in autologous hematopoietic stem cells is a potentially powerful approach to treat β-thalassemia. The challenge facing this approach is to stably express high levels of the human β-globin gene in an erythroid-specific, regulated, and sustained fashion. Low level expression, position effects and transcriptional silencing have hampered until now the effectiveness of viral transduction of the human β-globin gene linked to minimal regulatory sequences. I show here that the use of recombinant lentiviruses enables efficient transfer and faithful integration of the human β-globin gene together with large segments (3.2 kb) of its locus control region (LCR). Studies comparing a vector containing a 3.2 kb LCR, termed TNS9, to one with a minimal 1.0 kb LCR, termed RNS1, demonstrate both a higher mean level of human β-globin expression by TNS9, and a higher fraction of cells expressing human β-globin following vector integration of random sites. In long-term studies in recipient mice engrafted with TNS9-transduced bone marrow cells, production of lentivirus-encoded β-globin is substantially augmented, owing to an increase in both the level of globin expression, as shown by RNA analysis, and the fraction of red cells expressing human β A, as demonstrated by immunostaining. Murine α₂ : human βA₂ tetramers account for up to 13% of total hemoglobin in mature red cells in normal long-term bone marrow chimeras. Most importantly, higher levels are obtained in β-thalassemic mice, ranging from 17 to 24% fifteen weeks after transplant, resulting in a substantial increase in hemoglobin concentration and hematocrit levels, with a concomitant reduction in reticulocyte counts. Red cell morphology (anisocytosis and poikilocytosis) is markedly corrected. Therapeutic benefits are stable up to 40 weeks resulting in reduced extramedullary erythropoiesis in the spleen. My findings demonstrate that high-level, tissue-specific gene expression can be achieved in the progeny of unselected genetically modified stem cells. Therefore, a genetic approach could be successful in treating β-thalassemia. Furthermore, these findings provide a paradigm for stem cell therapy requiring regulated expression of a tissue-specific transgene in the progeny of genetically modified stem cells.