The major goal of this proposal is to develop and test novel, genetic methods for correcting sickle cell disease. The protocols will initially be tested in a transgenic mouse model. We recently produced adult mice that synthesize 99% human HbS and 1% human HbF; no mouse hemoglobin is produced in these adult animals. The mice are anemic and 10% of erythrocytes in peripheral blood are sickled. Preliminary studies suggest significant in vivo pathology; however, the animals are viable and fertile. We plan to examine these animals in longitudinal studies to evaluate the progression of the disease. When the HbS animals are fully characterized, hematopoietic stem cells will be purified from these mice and infected with recombinant AAV (Adeno-Associated Viral) and retroviral stocks that contain anti-sickling (beta/AS) globin genes. These genes are designed to effectively inhibit HbS polymerization and, therefore, to inhibit erythrocyte sickling. When conditions required for efficient transduction of stem cells are defined, purified stem cells from HbS mice will be infected and transplanted into the mouse model. These mice will then be evaluated to determine whether in vivo pathology is reduced or eliminated. An alternative genetic therapy for sickle cell disease will also be developed. A modified transcription factor (Erythroid Krupple Like Factor; EKLF) that binds to and activates the delta-globin gene will be designed. Transduction of this factor into transgenic mice that contain human delta-and beta/s- globin genes or into human hematopoietic stem cells will stimulate expression of the delta-globin gene which has powerful anti-sickling properties. The advantage of this system is that relatively low levels of transcription factor expression can simulate relatively high levels of delta-globin gene expression. A major impediment to successful genetic therapy has been the suppression of gene expression in virally transduced cells. In many cases, expression is high immediately after genes are transformed but synthesis is subsequently suppressed and sometimes completely silenced. We recently demonstrated that sodium butyrate and trichostatin A dramatically reactivate silenced, virally transduced genes. We propose to test these drugs for reactivation of beta/AS-globin and delta-EKLF genes after viral transduction and transplantation of hematopoietic stem cells. When the protocols described above are proven safe and effective in the transgenic mouse model and in isolated human hematopoietic stem ells, we plan to evaluate thee methods in human clinical trials.
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