Sustained fetal hemoglobin induction in vivo is achieved by BCL11A interference and coexpressed truncated erythropoietin receptor

Abstract

In xenograft mice and nonhuman primates, BCL11A interference and coexpression of a truncated erythropoietin receptor led to sustained hemoglobin F. Improved gene therapy for SCD Sickle cell disease (SCD) leads to substantial morbidity and decreased life span. It has been proven difficult to achieve therapeutic γ-globin production using hematopoietic stem cell gene therapy. Uchida et al. addressed this by developing a truncated human erythropoietin receptor to increase erythroid cell proliferation and differentiation. They added a microRNA-adapted short hairpin RNA interfering with the gene BCL11A (shmiR BCL11A) to increase γ-globin concentrations. A lentiviral vector encoding shmiR BCL11A and the truncated erythropoietin receptor was injected into CD34+ cells of macaques and autologously transplanted, leading to increased erythroid cells producing γ-globin at therapeutic levels. This promising approach may lead to improved gene therapy for SCD. Hematopoietic stem cell gene therapy for hemoglobin disorders, including sickle cell disease, requires high-efficiency lentiviral gene transfer and robust therapeutic globin expression in erythroid cells. Erythropoietin is a key cytokine for erythroid proliferation and differentiation (erythropoiesis), and truncated human erythropoietin receptors (thEpoR) have been reported in familial polycythemia. We reasoned that coexpression of thEpoR could enhance the phenotypic effect of a therapeutic vector in erythroid cells in xenograft mouse and autologous nonhuman primate transplantation models. We generated thEpoR by deleting 40 amino acids from the carboxyl terminus, allowing for erythropoietin-dependent enhanced erythropoiesis of gene-modified cells. We then designed lentiviral vectors encoding both thEpoR and B cell lymphoma/leukemia 11A (BCL11A)–targeting microRNA-adapted short hairpin RNA (shmiR BCL11A) driven by an erythroid-specific promoter. thEpoR expression enhanced erythropoiesis among gene-modified cells in vitro. We then transplanted lentiviral vector gene-modified CD34+ cells with erythroid-specific expression of both thEpoR and shmiR BCL11A and compared to cells modified with shmiR BCL11A only. We found that thEpoR enhanced shmiR BCL11A–based fetal hemoglobin (HbF) induction in both xenograft mice and rhesus macaques, whereas HbF induction with shmiR BCL11A only was robust, yet transient. thEpoR/shmiR BCL11A coexpression allowed for sustained HbF induction at 20 to 25% in rhesus macaques for 4 to 8 months. In summary, we developed erythroid-specific thEpoR/shmiR BCL11A–expressing vectors, enhancing HbF induction in xenograft mice and rhesus macaques. The sustained HbF induction achieved by addition of thEpoR and shmiR BCL11A may represent a viable gene therapy strategy for hemoglobin disorders.