Engineering of the endogenous HBD promoter increases HbA2

The ss-hemoglobinopathies, such as sickle cell disease and ss-thalassemia, are one of the most common genetic diseases worldwide and are caused by mutations affecting the structure or production of ss-globin subunits in adult hemoglobin. Many gene editing efforts to treat the ss-hemoglobinopathies attempt to correct ss-globin mutations or increase.-globin for fetal hemoglobin production. delta-globin, the subunit of adult hemoglobin A2, has high homology to ss-globin and is already pan-cellularly expressed at low levels in adult red blood cells. However, upregulation of delta-globin is a relatively unexplored avenue to increase the amount of functional hemoglobin. Here, we use CRISPR-Cas9 to repair non-functional transcriptional elements in the endogenous promoter region of delta-globin to increase overall expression of adult hemoglobin 2 (HbA2). We find that insertion of a KLF1 site alone is insufficient to upregulate delta-globin. Instead, multiple transcription factor elements are necessary for robust upregulation of delta-globin from the endogenous locus. Promoter edited HUDEP-2 immortalized erythroid progenitor cells exhibit striking increases of HBD transcript, from less than 5% to over 20% of total ss-like globins in clonal populations. Edited CD34 +hematopoietic stem and progenitors (HSPCs) differentiated to primary human erythroblasts express up to 46% HBD in clonal populations. These findings add mechanistic insight to globin gene regulation and offer a new therapeutic avenue to treat ss-hemoglobinopathies.

Automated summary

The ss-hemoglobinopathies, such as sickle cell disease and ss-thalassemia, can be treated by increasing the expression of delta-globin through gene editing techniques. This offers a new therapeutic approach for these genetic diseases.