Induction of fetal hemoglobin: Lentiviral shRNA knockdown of HBS1L in β0-thalassemia/HbE erythroid cells

PLOS ONE RESEARCH ARTICLE Induction of fetal hemoglobin: Lentiviral shRNA knockdown of HBS1L in β0thalassemia/HbE erythroid cells Sukanya Chumchuen ID1☯, Orapan Sripichai2☯¤, Natee Jearawiriyapaisarn ID2, Suthat Fucharoen2, Chayanon Peerapittayamongkol ID1* a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 1 Department of Biochemistry, Faculty of Medicine Siriraj Hospital, Mahidol University, Bangkok, Thailand, 2 Thalassemia Research Center, Institute of Molecular Biosciences, Mahidol University, Nakhon Pathom, Thailand ☯ These authors contributed equally to this work. ¤ Current address: Department of Medical Sciences, National Institute of Health, Ministry of Public Health, Nonthaburi, Thailand * , Abstract OPEN ACCESS Citation: Chumchuen S, Sripichai O, Jearawiriyapaisarn N, Fucharoen S, Peerapittayamongkol C (2023) Induction of fetal hemoglobin: Lentiviral shRNA knockdown of HBS1L in β0-thalassemia/HbE erythroid cells. PLoS ONE 18(3): e0281059. https://doi.org/10.1371/ journal.pone.0281059 Editor: Michela Grosso, University of Naples Federico II, ITALY Received: November 10, 2021 Accepted: January 16, 2023 Published: March 8, 2023 Peer Review History: PLOS recognizes the benefits of transparency in the peer review process; therefore, we enable the publication of all of the content of peer review and author responses alongside final, published articles. The editorial history of this article is available here: https://doi.org/10.1371/journal.pone.0281059 Copyright: © 2023 Chumchuen et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Data Availability Statement: All relevant data are within the paper and its Supporting information files. Imbalanced globin chain output contributes to thalassemia pathophysiology. Hence, induction of fetal hemoglobin in β-thalassemia and other β-hemoglobinopathies are of continuing interest for therapeutic approaches. Genome-wide association studies have identified three common genetic loci: namely β-globin (HBB), an intergenic region between MYB and HBS1L, and BCL11A underlying quantitative fetal hemoglobin production. Here, we report that knockdown of HBS1L (all known variants) using shRNA in early erythroblast obtained from β0-thalassemia/HbE patients triggers an upregulation of γ-globin mRNA 1.69 folds. There is modest perturbation of red cell differentiation assessed by flow cytometry and morphology studies. The levels of α- and β-globin mRNAs are relatively unaltered. Knockdown of HBS1L also increases the percentage of fetal hemoglobin around 16.7 folds when compared to non-targeting shRNA. Targeting HBS1L is attractive because of the potent induction of fetal hemoglobin and the modest effect on cell differentiation. Introduction The most common hereditable anemia in Thailand is thalassemia. There are two major types of thalassemia, α- and β-. The disease forms of β-thalassemia in Southeast Asia occur by a combination of β0 with β+, and the most common form of β+ is hemoglobin E [1]. G to A substitution at codon 26 of β-globin transcript leads to glutamate replaced by lysine residues. This mutation also generates an abnormal splice site resulting in an aberrant RNA processing competing with the normal splice site of βE-globin mRNA culminating in a reduction in βE-globin mRNA production and makes hemoglobin E a mild form of β+-thalassemia [2]. The symptoms of β-thalassemia/HbE display a complex trait [3, 4]. There are numerous modifiers, for example, coinheritance with α-thalassemia [5], the number of α-globin genes, types of β-globin mutations [6], and modifiers of HbF levels [7]. The gold standard treatment PLOS ONE | https://doi.org/10.1371/journal.pone.0281059 March 8, 2023 1 / 14 PLOS ONE Funding: Yes. This work was supported by Siriraj Research Fund and National Research Council of Thailand (NRCT). Funders play no role in the study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing interests: The authors have declared that no competing interests exist. HBS1L knockdown and induction of fetal hemoglobin for β-thalassemia is an allogenic bone marrow transplantation. With several constraints such as applicable to the relatively young age patients and suitable HLA-matched donors make this curative approach unavailable for most β-thalassemia patients [8]. Alternatively, genetherapy approaches to elicit the induction of β- or γ-globins, can diminish the load of unpaired α-globin chains. This will reclaim the balance of α/β-like globin ratios and thereby amend the ineffective erythropoiesis and shorten RBC lifespan [9]. Transduction of stem cells with lentivirus carrying short hairpin sequence (shRNA) targets to BCL11A and SOX6 has been shown to effectively induce γ-globin mRNA expression [10, 11]. A genome-wide association study (GWAS) from a European twin cohort with significantly different F-cell values revealed that 10% of the variability is attributable to the HBB region, 19% in HBS1L-MYB intergenic region (HMIR), and 15% in BCL11A on chromosome 2 [12]. GWAS study in Thai β0-thalassemia/HbE patients exhibits an association of the same three regions with the disease severity and HbF levels [13]. In humans, HBS1L is identified as one of 5 protein-coding genes (AHI1, MYB, ALDH8A1, HBS1L, and PDE7B) in around 1.5 Mb region associated with HbF levels on the chromosome 6q23 [14]. HBS1L functions in combination with PELOTA in identifying the stalled ribosome complexes on truncated mRNAs and causing them to be separated into subunits [15]. Additionally, this incident starts mRNA degradation pathways. We previously indicated that an SNP in exon 1 of HBS1L was associated with fetal hemoglobin and disease severity in Thai Chinese β0-thalassemia/HbE patients [16]. Expression of HBS1L is significantly diminished in erythroblasts throughout Phase II culture obtained from individuals with hereditary persistent fetal hemoglobin (HPFH) [17]. Here we extended that suppression of HBS1L expression by shRNA triggered considerable induction of γ-globin expression in both mRNA and protein levels while did not affect both α- and β-globin expression. Moreover, unlike any HbF inducer known so far, HbF’s induction slightly perturbed erythroid differentiation characterized by flow cytometry. Materials and methods Subjects This study was permitted by the Ethical Committee, Siriraj Institutional Review Board (Si 290/ 2015). All participants aged greater than 18 years old. The 5 β0-thalassemia/HbE patients were recruited from the Thalassemia clinic, Nakhon Pathom Hospital, Thailand, and the 5 healthy volunteers were recruited at the Institute of Molecular Bioscience, Mahidol University, Thailand (S1 Table). The β0-thalassemia/HbE patients did not receive blood transfusion for at least 1 month befo (...truncated)