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.
All data generated are included in the manuscript. All DNA sequences and oligo information are listed in Supplemental Table 1.
- Mandy Y Boontanrart
- Mandy Y Boontanrart
The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.
- Kate Quinlan, UNSW Sydney, Australia
© 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.
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.
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.