Highly efficient therapeutic genome editing of hematopoietic stem and progenitor cells to cure sickle cell disease and thalassemia
Sickle cell disease and β-thalassemia are globally the most common monogenic diseases. The common feature is mutations within the beta-globin gene leading to aberrant expression or protein function. Allogeneic stem cell transplantation is currently the only potential curative option, however, donor matching as well as post transplantation complications significantly hamper the success rate of this therapy. With the most recent developments in the CRISPR/Cas9 field we aim to develop a genome editing strategy on HSPCs that allow for autologous stem cell transplantations for both hemoglobinopathies. Our objective is to re-express gamma-globin(g) at the expense of the mutated beta-globin. Gamma-globin is normally only expressed during fetal development and the first 1-3 years after birth. It is widely accepted that 25% fetal hemoglobin (g2a2) expression in erythrocytes is sufficient for patients to remain symptom free as indicated by asymptomatic individuals with beta globin mutations but with concomitant high levels of fetal hemoglobin. Gamma-globin expression is controlled by the master regulator BCL11A. However, downregulation of this gene should be performed with great precision since it also exerts a critical role in B-cell development and hematopoietic stem cells homeostasis.
We and others have identified intronic erythroid specific enhancer elements in BCL11A using ATAC sequencing to which multiple guides where developed disrupting specific element regions. We have setup a viral free highly optimized CRISPR/Cas9 ribonucleoprotein (RNP) nucleofection that allows for knockout efficiencies up to 80% in human HSPCs. Furthermore, we have developed a novel transfection buffer that results in reduced toxicity for HSPCs.
Deletion of various regions within the BCL11A erythroid specific enhancer element lead to variable induction of fetal hemoglobin expression, up to 60%, that could be correlated to the specific transcription factors bound to the respective edited regions. Within he HSPC population no difference in genome editing efficiency was observed while furthermore, genome editing lead to limited effects on HSPC cell survival or erythroblast proliferation and differentiation.
Making modifications in the BCL11A enhancer results in fetal hemoglobin levels that exceed the minimum clinical limit to cure SCD and thalassemia. In general, we show that mutations created in HSPCs are stable and we do not observe major toxic effects. Targeting specific sites that are occupied by erythroid restrictive transcriptional regulators may prevent collateral BCL11A knockout in other lineages. These data pave the road towards transplanting genome edited HSPCs to allow for autologous stem cell transplantations and thereby bypass current limitations.