The developmental switch from production of gamma to beta globin chains normally follows a fixed schedule, accelerating at 32-34 weeks' gestation and reaching completion 16-20 weeks after birth. This switch is delayed in infants of diabetic mothers (IDM). Our preliminary results suggest that increased plasma concentrations of alpha amino-n-butyric acid and insulin in IDM augment gamma globin and inhibit beta globin expression. The hypothesis to be tested in this proposal is that butyrate analogues administered in the perinatal period have potential therapeutic value for delaying the gamma to beta globin switch in patients with beta chain hemoglobinopathies and thalassemias. To test this hypothesis, an in vivo system - a fetal ovine model - and an in vitro system - erythroid progenitors from infants with beta globin chain disorders-will be used. The duration of efficacy of butyrate analogues in augmenting gamma globin expression will be determined. Since our preliminary data strongly suggest that critical concentrations of alpha-amino-n-butyric acid and insulin in the IDM delay the gamma to beta switch, mechanisms of action underlying this effect will be explored in control and butyrate- treated erythroid progenitors by examining DNase sensitivity, histone acetylation, methylation patterns, nuclear run-offs, and cAMP levels. Transfection experiments with globin gene promoter and coding sequences linked to a reporter (neo) gene will be performed to identify potential sites of action or control regions in and surrounding the gamma delta beta complex that respond to butyrate. Since the half-life of butyric acid is short, new analogues will be synthesized and tested for efficacy and duration of action in both in vitro and in vivo systems. The longe range goal is to confirm that butyrate, either alone or in synergy with other plasma factors in IDM, delays the gamma to beta globin gene switch and to determine if butyrate has therapeutic value for preventing the gamma to beta globin switching process in patients afflicted with beta globin chain disorders.
