Mechanisms of Cis-Acting HbF Regulation in Sickle Cell Anemia

A therapeutic goal in sickle cell anemia is to increase HbF concentration to protect erythrocytes from sickle polymer-induced injury. HbF gene expression is regulated by cis-and trans-acting loci. We are focusing on novel cis-acting loci that were associated with elevated HbF in the Arab-Indian (AI) haplotype of sickle cell anemia. Our central hypothesis is that variants in the β-globin gene LCR in sickle cell anemia with the AI haplotype modulate HbF gene expression by differential looping to globin gene promoters and account, in part, for the variance in HbF levels associated with different HBB haplotypes. Using genetically modified sickle cell disease haplotype-specific iPSCs we are assessing the effects of putative cis-acting regulators of HbF expression. We will use gene editing to target selected cis-acting elements to modulate HbF gene expression in adult-type erythroblasts derived from sickle cell anemia patients with the high HbF AI and low HbF Benin HBB gene cluster haplotypes. We will functionally validate novel cis-acting HbF modulators using the directed differentiation of sickle cell disease-specific iPSCs into fetal and adult-type blood cells. Directed differentiation of sickle iPSCs into fetal and adult hemoglobin-expressing erythroblasts will allow us to explore the cell intrinsic effects of modified regulatory regions. Furthermore we are studying the effects of cis-activating HbF modulators in iPSC-derived erythroid cells using chromosome conformation capture, chromatin immunoprecipitation and global transcriptional analysis.