In Vivo Activation of the δ Globin Gene Promoter.

Abstract

The δ globin gene is the second adult β-like globin gene in humans and codes for the δ globin chain which forms together with the α globin chain Hemoglobin A2 (HbA2). HbA2 represents less than 3% of the total hemoglobin in normal individuals and it is typically increased in β thalassemia carriers. The δ globin gene is highly homologous to the β globin gene since it derives from a common ancestor.In our previous work we (as well as others) have demonstrated, in vitro, that the creation of the β globin proximal CACCC box consensus sequence, the binding site of the trascription factor EKLF, on the δ globin gene promoter is sufficient to enhance its expression to a considerable extent. Here we show that the δ globin gene promoter can be activated “ in vivo” in a transgenic mice model. We have produced transgenic mice lines with a DNA construct in which the wild type (wt) β globin gene promoter and either the wt or the proximal CACCC box containing δ globin gene promoter are linked in cis to the second hypersensitive site (HS2) of the Locus Control Region (LCR). The order of the different elements in our construct mimic the organization of the human β globin cluster were the δ globin gene is situated 5′ to the β and relatively closer to the LCR. The δ and β globin gene promoter are respectively linked to two different luciferase reporter genes, fireflies and renilla. All the transgenic lines produced are multicopy as assessed by southern blotting. The level of the expression of the two reporter genes has been assessed in the fetal liver at day 14 post coitum (pc). We have analyzed 3 out of 5 independent transgenic lines bearing the CACCC containing δ globin gene promoter construct, and 2 independent lines bearing the control construct (wt δ globin gene promoter). For each transgenic line we have analyzed two different litters. Transgenic fetuses have been identified by PCR. Fetal liver samples have been lisated and the crude protein extracted has been assayed for the luciferase versus renilla activity. Taking into account the different activity of the two reporter enzymes the wt δ globin gene promoter activity is 13% (+/−0,5) that of the wt β globin gene promoter in fetal liver (n = 18). On the other hand the transgenic lines bearing the construct with the CACCC box containing δ globin gene promoter show an activation up to 82% (+/−19) in respect to the wt β promoter, on average (n = 32) The difference in expression between the wt and the CACCC box bearing δ globin gene promoter is highly significant (ttest = 3.8X10−7 ). This observation is a step forwards to possible gene therapy strategies for hemoglobinopathies based on the reactivation of the endogenous δ globin gene. The δ globin chain could represent in fact a valid substituted of the δ globin chain in beta thalassemia patients. It is also well known that HbA2 is a powerful antisickling agent which could benefit sickle cell patients. Current gene therapy strategies for the hemoglobinopathies focus on the trasduction of the hematopoietic stem cells by viral vectors or on the correction of the endogenous β globin gene by homologous recombination. An alternative approach could be to introduce into the hematopoietic stem cells of an engineered transcription factor able to enhance δ globin gene transcription.