Krüppel-like factor 1(KLF1) is a hematopoietic-specific zinc finger transcription factor essential for erythroid gene expression. In concert with the transacting factor GATA1, KLF1 modulates the coordinate expression of the genes encoding the multi-enzyme heme biosynthetic pathway during erythroid differentiation. To explore the mechanisms underpinning KLF1 action at the gene loci regulating the first 3 steps in this process, we have exploited the K1-ERp erythroid cell line, in which KLF1 translocates rapidly to the nucleus in response to treatment with 4-OH-Tamoxifen (4-OHT). KLF1 acts as a differentiation-independent transcriptional co-regulator of delta-aminolevulinic acid dehydratase (Alad), but not 5-aminolevulinate synthase gene (Alas2) or porphobilinogen deaminase (Pbgd). Similar to its role at the β-globin promoter, KLF1 induces factor recruitment and chromatin changes at the Alad1b promoter in a temporally-specific manner. In contrast to these changes, we observed a distinct mechanism of histone eviction at the Alad1b promoter. Furthermore, KLF1-dependent events were not modulated by GATA1 factor promoter co-occupancy alone. These results not only enhance our understanding of erythroid-specific modulation of heme biosynthetic regulation by KLF1, but provide a model that will facilitate the elucidation of novel KLF1-dependent events at erythroid gene loci that are independent of GATA1 activity.
In developing countries, diagnostic tests for homozygous (HbSS) or compound heterozygous (HbSC or HbS-Beta thalassemia) sickle cell disease (SCD) are not readily available at the point-of-care (POC). Very few infants are screened in Africa for SCD because of the high cost and level of skill needed to run traditional tests. Current methods are too costly and take too much time to enable equitable and timely diagnosis to save lives. The World Health Organization recognizes a crucial need for early detection of SCD in newborns, since it is estimated that 70% SCD-related deaths in Africa are preventable with early cost-effective interventions. The diagnostic barrier can be broken with affordable, POC tools that facilitate early detection immediately after birth. We have developed a mobile micro-electrophoretic device (HemeChip) through which to quickly, accurately, and affordably screen for SCD (Fig. 1A). The HemeChip uses a microfabricated platform housing cellulose acetate electrophoresis to rapidly separate hemoglobin (Hb) types. Less than 5 microliters of blood, which can be obtained through a finger stick or heel stick, is processed on a piece of cellulose paper in alkaline buffer. The HemeChip reliably identifies and discriminates amongst Hb C/A2, S, F and A0. The micro-electrophoresis results were validated against standard clinical hemoglobin screening methods, including high performance liquid chromatography (HPLC), with Pearson Correlation Coefficient (PCC) of ≥0.96 relative to HPLC for all Hb types tested. The receiver Operating-Characteristic (ROC) curves showed more than 0.89 sensitivity and 0.86 specificity for identification of hemoglobin types using the HemeChip, based on the travelling distance from the sample application point (Fig. 1B). We developed a web-based image processing application for automated and objective quantification of HemeChip results at the POC using cloud computing resources (Fig. 1C). This intensity-based mobile phone image quantitation method showed high correlation with HPLC results for tested patient blood samples (PCC=0.95). HemeChip can distinguish between different patient phenotypes, including HbSS (HbS only), transfused HbSS (HbS and HbA), and Hemoglobin SC disease (HbS and HbC). In conclusion, the HemeChip identification and quantification of hemoglobin phenotypes, as a POC technique, were comparable to standard clinical methods. This platform has clinical potential in under-served populations worldwide, in which SCD is endemic. Figure 1. Mobile micro-electrophoretic device (HemeChip) for point-of-care screening for sickle cell disease. ( A) HemeChip prototype is shown with a miniscule blood sample that has been separated into characteristic hemoglobin bands. (B) The receiver Operating-Characteristic (ROC) curves show sensitivity and specificity of HemeChip for differentiating between adjacent hemoglobin bands based on the travelling distance from the sample application point. band traveling distance thresholds are shown: circle=7.5 mm, triangle=10.0 mm, and square=12.5 mm. (C) Web-based image processing application for automated and objective quantification of HemeChip results at the POC using cloud computing resources. Figure 1. Mobile micro-electrophoretic device (HemeChip) for point-of-care screening for sickle cell disease. ( A) HemeChip prototype is shown with a miniscule blood sample that has been separated into characteristic hemoglobin bands. (B) The receiver Operating-Characteristic (ROC) curves show sensitivity and specificity of HemeChip for differentiating between adjacent hemoglobin bands based on the travelling distance from the sample application point. band traveling distance thresholds are shown: circle=7.5 mm, triangle=10.0 mm, and square=12.5 mm. (C) Web-based image processing application for automated and objective quantification of HemeChip results at the POC using cloud computing resources. Disclosures No relevant conflicts of interest to declare.
Gene editing the BCL11A erythroid enhancer is a validated approach to fetal hemoglobin (HbF) induction for β-hemoglobinopathy therapy, though heterogeneity in edit allele distribution and HbF response may impact its safety and efficacy. Here we compared combined CRISPR-Cas9 endonuclease editing of the BCL11A +58 and +55 enhancers with leading gene modification approaches under clinical investigation. We found that combined targeting of the BCL11A +58 and +55 enhancers with 3xNLS-SpCas9 and two sgRNAs resulted in superior HbF induction, including in engrafting erythroid cells from sickle cell disease (SCD) patient xenografts, attributable to simultaneous disruption of core half E-box/GATA motifs at both enhancers. We corroborated prior observations that double strand breaks (DSBs) could produce unintended on-target outcomes in hematopoietic stem and progenitor cells (HSPCs) such as long deletions and centromere-distal chromosome fragment loss. We show these unintended outcomes are a byproduct of cellular proliferation stimulated by ex vivo culture. Editing HSPCs without cytokine culture bypassed long deletion and micronuclei formation while preserving efficient on-target editing and engraftment function. These results indicate that nuclease editing of quiescent hematopoietic stem cells (HSCs) limits DSB genotoxicity while maintaining therapeutic potency and encourages efforts for in vivo delivery of nucleases to HSCs.
Human induced pluripotent stem cells (hiPSCs) hold remarkable capacity for disease modeling and the development of novel therapeutic treatments for sickle cell disease (SCD). hiPSCs can theoretically produce all cell types including induced red blood cells (iRBCs). Sickle cell patients, in particular, could benefit from autologous, engineered red blood cells (RBCs). Many patients possess rare Rh phenotypes, are allo-sensitized to blood products and are at risk of iron overload from recurrent transfusions. Therefore, the generation of personalized iRBCs is attractive. Yet, in vitro iRBC production has been hampered by an inability of these cells to differentiate into terminally-mature, enucleated, beta globin-expressing RBCs. Here, we describe updated strategies to improve in vitro production of iRBCs. hiPSCs from sickle cell patients as well as those with normal hemoglobin were differentiated into hematopoietic stem progenitor cells (HSPCs) and immortalized via the overexpression of a previously characterized set of transcription factors promoting self-renewal and multipotency under the control of a doxycycline-regulated promoter. Utilizing an in vitro protocol incorporating increasing concentrations of human plasma, HSPCs differentiated from these lines proceed through terminal erythroid differentiation, including the formation of CD71-/GlyA+/α4 integrin-/Band 3+ cells. Plasma-stimulated iRBCs achieved robust enucleation (11-60.7%) and underwent fetal to adult globin-switching. Further, nearly 21% of the enucleated iRBCs were RNA negative erythrocytes 5-8 microns in diameter. RNA sequencing analysis reveals similar transcriptional profiles between iRBCs and peripheral blood CD34+- derived cultured RBCs (cRBCs) yet distinct differences between SCD and WT iRBCs. SCA iRBCs have increased extracellular matrix organization, cell-cell adhesive properties and up-regulation of hypoxia-response genes. Heme metabolism, DNA repair, fatty acid metabolism and oxidative phosphorylation are all impaired in SCD iRBCs. Assessment of cell physiology exposes membrane damage in SCD iRBCs with increased phalloidin permeability in comparison to wild type controls. Intriguingly, SCD iRBCs co-expressing gamma and beta-globin also demonstrate sickling under hypoxic conditions. With the development of expandable source of erythroid progenitors capable of producing mature red cells, we now aim to utilize this platform for robust disease modeling and autologous cell therapy. Figure. Figure. Disclosures Heeney: Pfizer: Research Funding; Sancilio Pharmaceuticals: Consultancy, Research Funding; Astra Zeneca: Consultancy, Research Funding; Ironwood: Research Funding; Novartis: Membership on an entity's Board of Directors or advisory committees; Vertex/Crisper: Other: Data Monitoring Committee.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.
hi@scite.ai
10624 S. Eastern Ave., Ste. A-614
Henderson, NV 89052, USA
Copyright © 2024 scite LLC. All rights reserved.
Made with 💙 for researchers
Part of the Research Solutions Family.