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bioRxiv · 10.1101/2022.12.19.521003

Engineering of the Endogenous HBD promoter increases HbA2

Boontanrart, M. Y.·Mächler, E.·Ponta, S.·Nelis, J. C.·Preiano, V. G.·Corn, J. E.
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Abstract

The {beta}-hemoglobinopathies, such as sickle cell disease and {beta}-thalassemia, are one of the most common genetic diseases worldwide and are caused by mutations affecting the structure or production of {beta}-globin subunits in adult hemoglobin. Many gene editing efforts to treat the {beta}-hemoglobinopathies attempt to correct {beta}-globin mutations or increase {gamma}-globin for fetal hemoglobin production. {delta}-globin, the subunit of adult hemoglobin A2, has high homology to {beta}-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 {beta}-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 {beta}-hemoglobinopathies.

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Boontanrart, M. Y., Mächler, E., Ponta, S., Nelis, J. C., Preiano, V. G., Corn, J. E.. 2022-12-20. Engineering of the Endogenous HBD promoter increases HbA2. https://doi.org/10.1101/2022.12.19.521003

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