Attivazione del gene delta globinico in vivo attraverso la creazione di topi transgenici quale modello di studio per la cura della beta talassemia

The δ globin gene is the minor adult β-like globin gene in humans. The δ globin chain forms together with the α globin chain, the 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 δ globin gene deriving from a common ancestor . In our previous work we ( as well as others) have demonstrated, in vitro, that the creation of the CACCC box consensus sequence on the globin gene promoter is sufficient to enhance its expression to a considerable extent. This observation is at the basis of 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 substitution 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. Here we show that: 1) The δ globin gene promoter can be activated in vivo in a transgenic mouse model; 2) The activated δ globin gene promoter is able to drive the expression of δ globin gene to a high level; 3) The crossing of the homozygous transgenic line LCR-δ CACCC with the heterozygous mouse model of β0-thalassemia (Hbbth-3/+) increased up to 3 g/dl the total hemoglobin level. First, we have produced transgenic mice lines with a DNA construct in which the wild type (wt) β globin gene promoter and the proximal CACCC box containing δ globin gene promoter are linked in cis to a single erythroid specific enhancer, the second hypersensitive site ( HS2) of the Locus Control Region (LCR), (HS2δMFLβRL). The order of the different elements in our construct mimics the spatial organization of the βglobin cluster were the δ globin gene is situated 5’ to the β gene and relatively closer to the LCR. We have also produced transgenic mice lines bearing the wtδ globin gene promoter on place of the CACCC containing δ, as a control line (HS2δ wtFLβRL). The δ and β globin gene promoters are respectively linked to two different (fireflies and renilla) luciferase reporter genes. The level of the expression of the two reporter genes has been assessed in the fetal liver at day 10.5, 12.5, 14.5 and 16.5 post coitum (pc). We have analyzed 3 independent transgenic lines bearing the CACCC containing δ globin gene promoter construct(HS2δ FLβ RL), and 3 independent lines bearing the control construct (HS2δwtFLβ RL). For each transgenic line we have analyzed 3 different pregnancies. Fetal liver samples have been lysated and the crude protein extracted has been assayed for the luciferase versus renilla activity. The expression level of the transgenic lines bearing the construct with the CACCC box containing δ globin gene promoter (HS2δ FLRβ L) show an activity greater than the control wt (HS2δ wtFLβ RL): 12.5pc (85.07%±15,8 – t test 1.57164E-06), 14.5pc (78,74%±22,6 – t test 0.0002) e 16.5pc (97,35%±29,3 – t test 0.0006. After these preliminary results, we have produced a transgenic line carrying the mini LCR (HS- HS4) and the δ gene driven by the CACCC containing δ promoter (CACCCδM-LCR). Here we show that the δ globin gene can be activated in vivo reaching high levels of expression in our transgenic mouse model. Our results on a single copy transgenic line show an expression level of the δ gene of 30% compared to the endogenous beta major. This level of expression could be considered curative for beta thalassemia and sickle cell disease. After this result, we decided to intercross the homozygous transgenic line CACCCδM-LCR with the heterozygous mouse model of beta-thalassemia (Hbb-th3/+). Hematological analysis performed at 8 weeks after-born mice Hbb-th3/+ revealed 8,7±1,2 g/dL of total hemoglobin levels versus 10,5 g/dL ± 1,3 in heterozygous CACCCδM-LCR /Hbb-th3/+ mice, 11,6±0,94 g/dL in homozygous CACCCδM-LCR /Hbb-th3/+ and 15.48±1.4 g/dL in wild type mice. We plan to obtain the final validation of the δ globin gene as a therapeutic gene by the rescue of the thalassemic mouse model th3/th3. Current gene therapy strategies for the hemoglobinopathies focus on the trasduction of the hematopoietic stem cells by viral vectors. More recently strategies involving the correction of the endogenous δ globin gene by homologous recombination have been proposed. An alternative approach could be to introduce into the hematopoietic stem cells an engineered transcription factor able to enhance δ globin gene transcription. Recently enormous developments have been made in engineering sequence specific zinc finger DNA-binding protein. It is thus conceivable to design a δ specific transcription factor able to specifically activate the endogenous δ globin gene as a therapeuthical tool for the hemoglobinopathies.