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

  1. Mandy Y Boontanrart

    Department of Biology, ETH Zurich, Zurich, Switzerland
    For correspondence
    mandy.boontanrart@biol.ethz.ch
    Competing interests
    The authors declare that no competing interests exist.
  2. Elia Mächler

    Department of Biology, ETH Zurich, Zurich, Switzerland
    Competing interests
    The authors declare that no competing interests exist.
  3. Simone Ponta

    Department of Biology, ETH Zurich, Zurich, Switzerland
    Competing interests
    The authors declare that no competing interests exist.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0002-0007-0346
  4. Jan C Nelis

    Department of Biology, ETH Zurich, Zurich, Switzerland
    Competing interests
    The authors declare that no competing interests exist.
  5. Viviana G Preiano

    Department of Biology, ETH Zurich, Zurich, Switzerland
    Competing interests
    The authors declare that no competing interests exist.
  6. Jacob E Corn

    Department of Biology, ETH Zurich, Zurich, Switzerland
    For correspondence
    jacob.corn@biol.ethz.ch
    Competing interests
    The authors declare that no competing interests exist.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0002-7798-5309

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

  1. Kate Quinlan, UNSW Sydney, Australia

Version history

  1. Received: November 29, 2022
  2. Preprint posted: December 20, 2022 (view preprint)
  3. Accepted: May 27, 2023
  4. Accepted Manuscript published: June 2, 2023 (version 1)
  5. 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.

Metrics

  • 942
    Page views
  • 165
    Downloads
  • 0
    Citations

Article citation count generated by polling the highest count across the following sources: Crossref, PubMed Central, Scopus.

Download links

A two-part list of links to download the article, or parts of the article, in various formats.

Downloads (link to download the article as PDF)

Open citations (links to open the citations from this article in various online reference manager services)

Cite this article (links to download the citations from this article in formats compatible with various reference manager tools)

  1. Mandy Y Boontanrart
  2. Elia Mächler
  3. Simone Ponta
  4. Jan C Nelis
  5. Viviana G Preiano
  6. Jacob E Corn
(2023)
Engineering of the endogenous HBD promoter increases HbA2
eLife 12:e85258.
https://doi.org/10.7554/eLife.85258

Further reading

    1. Genetics and Genomics
    2. Neuroscience
    Yoshifumi Sonobe, Soojin Lee ... Paschalis Kratsios
    Research Article Updated

    A hexanucleotide repeat expansion in C9ORF72 is the most common genetic cause of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). A hallmark of ALS/FTD pathology is the presence of dipeptide repeat (DPR) proteins, produced from both sense GGGGCC (poly-GA, poly-GP, poly-GR) and antisense CCCCGG (poly-PR, poly-PG, poly-PA) transcripts. Translation of sense DPRs, such as poly-GA and poly-GR, depends on non-canonical (non-AUG) initiation codons. Here, we provide evidence for canonical AUG-dependent translation of two antisense DPRs, poly-PR and poly-PG. A single AUG is required for synthesis of poly-PR, one of the most toxic DPRs. Unexpectedly, we found redundancy between three AUG codons necessary for poly-PG translation. Further, the eukaryotic translation initiation factor 2D (EIF2D), which was previously implicated in sense DPR synthesis, is not required for AUG-dependent poly-PR or poly-PG translation, suggesting that distinct translation initiation factors control DPR synthesis from sense and antisense transcripts. Our findings on DPR synthesis from the C9ORF72 locus may be broadly applicable to many other nucleotide repeat expansion disorders.

    1. Ecology
    2. Genetics and Genomics
    Franziska Grathwol, Christian Roos ... Gisela H Kopp
    Research Advance

    Adulis, located on the Red Sea coast in present-day Eritrea, was a bustling trading centre between the first and seventh centuries CE. Several classical geographers--Agatharchides of Cnidus, Pliny the Elder, Strabo-noted the value of Adulis to Greco--Roman Egypt, particularly as an emporium for living animals, including baboons (Papio spp.). Though fragmentary, these accounts predict the Adulite origins of mummified baboons in Ptolemaic catacombs, while inviting questions on the geoprovenance of older (Late Period) baboons recovered from Gabbanat el-Qurud ('Valley of the Monkeys'), Egypt. Dated to ca. 800-540 BCE, these animals could extend the antiquity of Egyptian-Adulite trade by as much as five centuries. Previously, Dominy et al. (2020) used stable istope analysis to show that two New Kingdom specimens of P. hamadryas originate from the Horn of Africa. Here, we report the complete mitochondrial genomes from a mummified baboon from Gabbanat el-Qurud and 14 museum specimens with known provenance together with published georeferenced mitochondrial sequence data. Phylogenetic assignment connects the mummified baboon to modern populations of Papio hamadryas in Eritrea, Ethiopia, and eastern Sudan. This result, assuming geographical stability of phylogenetic clades, corroborates Greco-Roman historiographies by pointing toward present-day Eritrea, and by extension Adulis, as a source of baboons for Late Period Egyptians. It also establishes geographic continuity with baboons from the fabled Land of Punt (Dominy et al., 2020), giving weight to speculation that Punt and Adulis were essentially the same trading centres separated by a thousand years of history.