Hereditary hemoglobinopathies have long been favorites of gene therapists, and a genetic cure would be a wonderful extension of the decades of research spent understanding the molecular biology of hemoglobin gene expression. The most realistic approaches rely on retroviral vectors, which have the potential to integrate in hematopoietic stem cells, albeit at disappointingly low frequencies in primates. In order to achieve high-level, erythroid-specific expression, scientists have turned to the β-globin locus for transcriptional control elements, especially globin promoters in combination with parts of the locus control region (LCR) and intron sequences. The low vector titers and frequent rearrangements caused by these elements highlight the difficulties associated with including sequences that are normally not transcribed, or are now transcribed in reverse orientation, in an RNA vector. Despite these problems, globin gene therapists have stubbornly persisted, and recent results show that optimized cassettes including β-globin LCR, promoter, and intron sequences can be incorporated into a lentiviral vector (instead of a conventional murine leukemia virus vector) and lead to potentially therapeutic hemoglobin protein levels in mice receiving transplants (May et al, Nature. 2000;406:82-86).
An alternative approach to globin vector design is to abandon problematic regulatory elements from the β-globin locus and achieve high-level, erythroid-specific expression some other way. Bodine and colleagues showed that a double-copy murine leukemia virus vector containing the promoter for the red cell membrane protein ankyrin produced a respectable 8% γ-globin transgene mRNA expression level (relative to endogenous α-globin) in mice receiving transplants (Sabatino et al, Proc Natl Acad Sci U S A. 2000;97:13294-13299). Here, Moreau-Gaudry and colleagues (page 2664) extend these findings by combining nonglobin, erythroid-specific promoters such as ankyrin with different erythroid enhancers and a viral posttranscriptional regulatory element in a self-inactivating lentiviral vector. Using a green fluorescent protein transgene, they find that the vectors can be produced at high titers, that they are stable, and that erythroid-specific expression can be obtained in a majority of erythroid cells in mice receiving transplants. A similar vector with a γ-globin transgene produced 11% to 28% mRNA expression levels (relative to endogenous α-globin) in MEL cells. Unfortunately, the globin vectors were not tested in transplantation experiments; so it is not clear if the same expression levels can be achieved in vivo. Still, these experiments demonstrate that the problems of including β-globin locus regulatory elements in retroviral vectors can be avoided, and there is no reason to believe that the optimal vector design has been achieved yet. The next test will be to see whether these vectors can surpass the impressive performance of lentiviral vectors based on the β-globin locus and produce therapeutic protein levels after transplantation.
