AN ALTERNATIVE APPROACH TO BETA THALASSEMIA THERAPY: HUMAN DELTA GLOBIN GENE ACTIVATION

The delta globin gene, located between the gamma and beta genes on human chromosome 11, produces a small amount of delta globin in children and adults, participating to the HbA2 synthesis. Our previous work showed in vitro that creation of the CACCC box consensus sequence on the delta globin gene promoter is sufficient to enhance its expression to a considerable extent. The delta globin chain could be a valid substitute of the beta globin chain in thalassemia disorder and an antisickling agent in sickle cell anemia. Here we show that: 1) the delta globin gene promoter can be activated in vivo in a transgenic mouse model; 2) the activated delta globin gene promoter is able to drive the expression of delta globin gene to a high level. We have produced transgenic mice lines with a DNA construct in which the wild type (wt) delta globin gene promoter and the proximal CACCC box containing delta globin gene promoter are linked in cis to a single erythroid specific enhancer. The order of the different elements in our construct mimics the spatial organization of the delta globin cluster were the delta globin gene is situated 5' to the beta gene and relatively closer to the LCR. We have also produced transgenic control lines, bearing the wt delta globin gene promoter on place of the CACCC containing delta. The delta and beta globin gene promoters are respectively linked to two different (firefly and renilla) luciferase reporter genes. We have analyzed 3 independent transgenic lines bearing the CACCC containing delta globin gene promoter construct, and 3 independent lines bearing the control construct (wt delta globin gene promoter). While the expression level of the wt delta globin gene promoter remains around 20% compared to the beta globin gene promoter in each time point observed (12.5, 14.5 and 16.5 days post coitum), the expression level of CACCC containing delta globin gene promoter reached high expression levels in fetal liver at 12.5 p.c. (82% ± 17), 14.5 pc (75% ± 23) and 16.5 pc (97% ± 29). We also have produced two transgenic lines carrying the mini LCR and the delta gene driven by the CACCC containing delta promoter (CACCCdeltaLCR). Our preliminary results on a single copy transgenic line show an expression level of the delta gene of 30% compared to the endogenous beta major. This level of expression could be considered curative both for beta thalassemia and sickle cell disease. We plan to obtain the final validation of the delta globin gene as a therapeutic gene by the rescue of a thalassemic mouse model (th3/th3).