PT  - JOURNAL ARTICLE
AU  - Boontanrart, Mandy Y.
AU  - Mächler, Elia
AU  - Ponta, Simone
AU  - Nelis, Jan C.
AU  - Preiano, Viviana G.
AU  - Corn, Jacob E.
TI  - Engineering of the Endogenous <em>HBD</em> promoter increases HbA2
AID  - 10.1101/2022.12.19.521003
DP  - 2022 Jan 01
TA  - bioRxiv
PG  - 2022.12.19.521003
4099  - http://biorxiv.org/content/early/2022/12/20/2022.12.19.521003.short
4100  - http://biorxiv.org/content/early/2022/12/20/2022.12.19.521003.full
AB  - The β-hemoglobinopathies, such as sickle cell disease and β-thalassemia, are one of the most common genetic diseases worldwide and are caused by mutations affecting the structure or production of β-globin subunits in adult hemoglobin. Many gene editing efforts to treat the β-hemoglobinopathies attempt to correct β-globin mutations or increase γ-globin for fetal hemoglobin production. δ-globin, the subunit of adult hemoglobin A2, has high homology to β-globin and is already pan-cellularly expressed at low levels in adult red blood cells. However, upregulation of δ-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 δ-globin to increase overall expression of adult hemoglobin 2 (HbA2). We find that insertion of a KLF1 site alone is insufficient to upregulate δ-globin. Instead, multiple transcription factor elements are necessary for robust upregulation of δ-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 β-like globins. Edited CD34+ hematopoietic stem and progenitors (HSPCs) differentiated to primary human erythroblasts express up to 35% HBD. These findings add mechanistic insight to globin gene regulation and offer a new therapeutic avenue to treat β-hemoglobinopathies.Competing Interest StatementThe authors have declared no competing interest.