A one-base therapeutic insertion in the HBG2 distal promoter reactivates γ-globin expression

Background: The reactivation of developmental silenced γ-globin genes (HBG1/2) has shown promise as a therapeutic strategy for improving symptoms of β-hemoglobinopathies. Currently, the focus of therapeutic targets is primarily on the major fetal hemoglobin suppressors, such as BCL11A and ZBTB7A and of their binding sites on the proximal HBG promoter. However, the role of the distal HBG promoter in regulating gene expression remains to be explored. Methods: We used CRISPR/Cas9 system to edit the distal HBG promoter. In vitro and in vivo assays, as well as engrafted NCG-Kit-V831M mice, were used for functional validation and mechanistic studies. Results: We discovered an insertion of nucleotide A (insA) between − 1368 and − 1369 bp upstream of the TSS in HBG2 resulting in remarkable increase in γ-globin expression in HUDEP-2 cells. We also observed elevated γ-globin expression in human CD34⁺ erythroid progenitor cells from healthy individuals and those with β-thalassemia when introducing insA mutation. Similarly, engrafted NCG-Kit-V831M mice showed increased γ-globin expression. Importantly, neither did insA have any off-target effects nor did it affect the maturation of erythroid cells. Furthermore, we found that the insA mutation created a binding site for the transcription activator FOXO3, which was activated by AMPK. Additionally, introducing insA specifically demethylated the − 162 CpG site on HBG promoter by reducing the enrichment of DNA methyltransferase 3 A (DNMT3A). At the same time, it activated histone modifications and RNA polymerase II (Pol II) in both distal and proximal HBG promoter and might inhibit the binding of BCL11A and ZBTB7A on -115 and − 200 sites on the HBG promoter respectively. In addition, combination of insA and the − 115 or -200 editing targets resulted in an amplify effect in reactivating γ-globin genes expression. Conclusions: Overall, we presented the preclinical data to support the role of insA on regulating γ-globin expression using CD34⁺ HSPC cells derived from healthy donors or patients with β-thalassemia, and subsequently engrafted mice. Our study suggests that introducing insA mutation leads to significantly boosted fetal globin levels and uncovers new safe therapeutic target or strategy for β-hemoglobinopathies.