Engineering of the endogenous HBD promoter increases HbA2
- ETH Zurich, Switzerland
Abstract
The b-hemoglobinopathies, such as sickle cell disease and b-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 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 β-hemoglobinopathies.
Data availability
All data generated are included in the manuscript. All DNA sequences and oligo information are listed in Supplemental Table 1.
Article and author information
Author details
Funding
ETH Zürich Foundation (ETH Pioneer Fellowship)
- Mandy Y Boontanrart
SNSF (BRIDGE Proof of Concept)
- Mandy Y Boontanrart
The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.
Reviewing Editor
- Kate Quinlan, UNSW Sydney, Australia
Version history
- Received: November 29, 2022
- Preprint posted: December 20, 2022 (view preprint)
- Accepted: May 27, 2023
- Accepted Manuscript published: June 2, 2023 (version 1)
- Version of Record published: June 15, 2023 (version 2)
Copyright
© 2023, Boontanrart et al.
This article is distributed under the terms of the Creative Commons Attribution License permitting unrestricted use and redistribution provided that the original author and source are credited.
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Engineering of the endogenous HBD promoter increases HbA2
eLife 12:e85258.
https://doi.org/10.7554/eLife.85258
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