Generation of a High-Titer Retroviral Vector Capable of Expressing High Levels of the Human β-Globin Gene

Retrovirus-mediated gene transfer into hematopoietic cells may provide a means of treating both inherited and acquired diseases involving hematopoietic cells. Implementation of this approach for disorders resulting from mutations affecting the β-globin gene (e.g., β-thalassemia and sickle cell anemia), however, has been hampered by the inability to generate recombinant viruses able to efficiently and faithfully transmit the necessary sequences for appropriate gene expression. We have addressed this problem by carefully examining the interactions between retroviral and β-globin gene sequences which affect vector transmission, stability, and expression. First, we examined the transmission properties of a large number of different recombinant proviral genomes which vary both in the precise nature of vector, β-globin structural gene, and locus control region (LCR) core sequences incorporated and in the placement and orientation of those sequences. Through this analysis, we identified one specific vector, termed Mβ6L, which carries both the human β-globin gene and core elements HS2, HS3, and HS4 from the LCR and faithfully transmits recombinant proviral sequences to cells with titers greater than 106per ml. Populations of murine erythroleukemia (MEL) cells transduced by this virus expressed levels of human β-globin transcript which, on a per gene copy basis, were 78% of the levels detected in an MEL-derived cell line, Hu11, which carries human chromosome 11, the site of the β-globin locus. Analysis of individual transduced MEL cell clones, however, indicated that, while expression was detected in every clone tested (n = 17), the levels of human β-globin treatment varied between 4% and 146% of the levels in Hu11. This clonal variation in expression levels suggests that small β-globin LCR sequences may not provide for as strict chromosomal position-independent expression of β-globin as previously suspected, at least in the context of retrovirus-mediated gene transfer.