In the past few years several groups have produced transgenic mice t hat synthesize high levels of human sickle hemoglobin (Hb S). When erythrocytes from these animals are deoxygenated, greater than 90% of the cells display the same characteristic sickled shapes as erythrocytes from humans with humans with sickle cell disease. Compared to controls the mice have decreased hematocrits, elevated reticulocyte counts, lower hemoglobin concentrations, and splenomegaly, which are all indications of the anemia associated with sickle cell disease in humans. Although the production of these mice was a major first step ina the development of a mouse model for sickle cell disease, deletion of the endogenous mouse globin genes is required to perfect the model. We have recently deleted the endogenous mouse (betamaj and betamin globin genes and paszty and Rubin deleted the endogenous mouse alpha1 and alpha 2 globin genes. After exchanging mice with Rubin's group, we have bred these animals to transgenic mouse lines and express high levels of human fetal (HbF) and adult (HbA) hemoglobin and to animals that express high levels of human fetal (HbF) and sickle (HbS) hemoglobin. The fetal gene in these animals is designed to switch off later than normal to provide the best change for HbS mouse survival. To date, we have obtained mice that express 99% human HbA and 1% human HbF in adults. No mouse hemoglobin is produced in adults and these animals are healthy and reproduce normally. The final breedings to obtain animals that switch from HbF to HbS are in progress and we expect to have surviving animals soon. In a second approach, the mouse alpha- and beta-globin genes will be replaced with human alpha- and betas-globin or alpha, gamma- and betas-globin genes to obtain mice that synthesize 100% sickle hemoglobin. This approach will provide more control over alpha-and beta-globin chain balance which is difficult to achieve with transgenic mice made by microinjection. When the best model is defined, hematopoietic stem cells will be purified from these animals and infected with recombinant best model is defined, hematopoietic stem cells will be purified from these animals and infected with recombinant AAV (Adeno-Associated Viral) stocks that carry anti-sickling (betaAS) globin genes. These genes are designed to effectively inhibit Hb S polymerization and, therefore, to inhibit erythrocyte sickling. Transduced stem cells will be transplanted into HbS recipient mice and the efficiency of infection and level of expression will be analyzed. When protocols that yield infection efficiencies of 75% and expression levels of 10-15 % of total beta-globin chains are achieved, erythrocyte sickling will be analyzed both in vitro and in vivo and effects on in vivo pathology will be assessed. Finally, an alternative genetic therapy for sickle cell disease will be developed. A modified transcription factor (Erythroid Krupple Like Fact or, EKLF) that binds to and activates the delta-globin gene will be designed. Transduction of this factor into hematopoietic stem cells will stimulate expression of the delta-globin which has powerful anti-sickling properties. Preliminary experiments suggest that delta-globin gene expression can be increased to 15% of total beta-like globin chains by this method. The advantage of this system is that relatively low levels of transcription factor expression can stimulate relatively high levels of delta-globin gene expression. Inhibition of in vivo pathology by either of the methods described above will demonstrate athe efficacy of these strategies and will provide a foundation for genetic therapy of sickle cell disease in humans.
