Inherited non-hemolytic anemia is a group of rare bone marrow disorders characterized by erythroid defects. Although concerted efforts have been made to explore the underlying pathogenetic mechanisms of these diseases, the understanding of the causative mutations are still incomplete. Here we identify in a diseased pedigree that a gain-of-function mutation in toll-like receptor 8 (TLR8) is implicated in inherited non-hemolytic anemia.
View Article and Find Full Text PDFβ-thalassemia/HbE results from mutations in the β-globin locus that impede the production of functional adult hemoglobin. Base editors (BEs) could facilitate the correction of the point mutations with minimal or no indel creation, but its efficiency and bystander editing for the correction of β-thalassemia mutations in coding and non-coding regions remains unexplored. Here, we screened BE variants in HUDEP-2 cells for their ability to correct a spectrum of β-thalassemia mutations that were integrated into the genome as fragments of .
View Article and Find Full Text PDFDNA double-strand breaks (DSBs) induced by gene-editing tools are primarily repaired through non-homologous end joining (NHEJ) or homology-directed repair (HDR) using synthetic DNA templates. However, error-prone NHEJ may result in unexpected indels at the targeted site. For most genetic disorders, precise HDR correction using exogenous homologous sequence is ideal.
View Article and Find Full Text PDFLarge-scale in vitro red blood cell (RBC) production has been attempted in recent years. Potential cell sources for RBC production include hematopoietic stem/progenitor cells, pluripotent stem cells, and immortalized erythroid progenitor cell lines, which can induce enucleated RBCs with characteristics such as oxygen-carrying capacity and deformability. A phase I clinical study of cultured RBCs produced from hematopoietic stem/progenitor cells has revealed a similar in vivo half-life between cultured and native RBCs.
View Article and Find Full Text PDFMany therapeutic proteins are expressed in Escherichia coli bacteria for the low cost and high yield obtained. However, these gram-negative bacteria also generate undesirable endotoxin byproducts such as lipopolysaccharides (LPS). These endotoxins can induce a human immune response and cause severe inflammation.
View Article and Find Full Text PDFReactivation of fetal hemoglobin (HbF) is a commonly adapted strategy to ameliorate β-hemoglobinopathies. However, the continued production of defective adult hemoglobin (HbA) limits HbF tetramer production affecting the therapeutic benefits. Here, we evaluated deletional hereditary persistence of fetal hemoglobin (HPFH) mutations and identified an 11-kb sequence, encompassing putative repressor region (PRR) to β-globin exon-1 (βE1), as the core deletion that ablates HbA and exhibits superior HbF production compared with HPFH or other well-established targets.
View Article and Find Full Text PDFBackground: The generation of immortalized erythroid progenitor cell lines capable of producing enough red blood cells (RBCs) for blood transfusion typically requires the overexpression of oncogenes in stem cells or progenitor cells to permanently proliferate immature cells. It is essential that any live oncogene-expressing cells are eliminated from the final RBC products for clinical use.
Study Design And Methods: It is believed that safety issues may be resolved by using a leukoreduction filter or by irradiating the final products, as is conventionally done in blood banks; however, this has never been proven to be effective.
Genome editing tools, such as CRISPR/Cas, TALE nucleases and, more recently, double-strand-break-independent editors, have been successfully used for gene therapy and reverse genetics. Among various challenges in the field, tolerable and efficient delivery of editors to target cells and sites, as well as independence from commercially available tools for flexibility and fast adoption of new editing technology are the most pressing. For many hematopoietic research applications, primary CD34 cells and the human umbilical cord-derived progenitor erythroid 2 (HUDEP-2) cell line are highly informative substrates and readily accessible for manipulation.
View Article and Find Full Text PDFBackground And Objectives: The RHAG blood group system contains five antigens: Duclos (RHAG001), Ol (RHAG002), DSLK (RHAG003), Kg (RHAG005) and SHER (RHAG006). Individuals who are DSLK-negative and Kg-positive have the same allele RHAG*01.-3, with a single-nucleotide variation (rs144305805), c.
View Article and Find Full Text PDFSickle cell anaemia (SCA) is one of the common autosomal recessive monogenic disorders, caused by a transverse point mutation (GAG > GTG) at the sixth codon of the beta-globin gene, which results in haemolytic anaemia due to the fragile RBCs. Recent progress in genome editing has gained attention for the therapeutic cure for SCA. Direct correction of SCA mutation by homology-directed repair relies on a double-strand break (DSB) at the target site and carries the risk of generating beta-thalassaemic mutations if the editing is not error-free.
View Article and Find Full Text PDFDiamond Blackfan anemia (DBA) is a rare congenital disease characterized by defective maturation of the erythroid progenitors in the bone marrow, for which treatment involves steroids, chronic transfusions, or hematopoietic stem cells transplantation. Diamond Blackfan anemia is caused by defective ribosome biogenesis due to heterozygous pathogenic variants in one of 19 ribosomal protein (RP) genes. The decreased number of functional ribosomes leads to the activation of pro-apoptotic pathways and to the reduced translation of key genes for erythropoiesis.
View Article and Find Full Text PDFErythropoiesis is a highly complex and sophisticated multistage process regulated by many transcription factors, as well as noncoding RNAs. Anthrax toxin receptor 1 (ANTXR1) is a type I transmembrane protein that binds the anthrax toxin ligands and mediates the entry of its toxic part into cells. It also functions as a receptor for the Protective antigen (PA) of anthrax toxin, and mediates the entry of Edema factor (EF) and Lethal factor (LF) into the cytoplasm of target cells and exerts their toxicity.
View Article and Find Full Text PDFReactivation of fetal hemoglobin (HbF, 22) alleviates clinical symptoms in patients with -thalassemia and sickle cell disease, although the regulatory mechanisms of -globin expression have not yet been fully elucidated. Recent studies found that interfering with the expression of the membrane protein ANTXR1 gene upregulated -globin levels. However, the exact mechanism by which ANTXR1 regulates -globin levels remains unclear.
View Article and Find Full Text PDFRibosomal protein dysfunction causes diverse human diseases, including Diamond-Blackfan anemia (DBA). Despite the universal need for ribosomes in all cell types, the mechanisms underlying ribosomopathies, which are characterized by tissue-specific defects, are still poorly understood. In the present study, we analyzed the transcriptomes of single purified erythroid progenitors isolated from the bone marrow of DBA patients.
View Article and Find Full Text PDFMolecular therapies and functional studies greatly benefit from spatial and temporal precision of genetic intervention. We therefore conceived and explored tag-activated microRNA (miRNA)-mediated endogene deactivation (TAMED) as a research tool and potential lineage-specific therapy. For proof of principle, we aimed to deactivate γ-globin repressor in erythroid cells by tagging the 3' untranslated region (UTR) of with miRNA recognition sites (MRSs) for the abundant erythromiR miR-451a.
View Article and Find Full Text PDFNaturally occurring point mutations in the promoter switch hemoglobin synthesis from defective adult beta-globin to fetal gamma-globin in sickle cell patients with hereditary persistence of fetal hemoglobin (HPFH) and ameliorate the clinical severity. Inspired by this natural phenomenon, we tiled the highly homologous proximal promoters using adenine and cytosine base editors that avoid the generation of large deletions and identified novel regulatory regions including a cluster at the -123 region. Base editing at -123 and -124 bp of promoter induced fetal hemoglobin (HbF) to a higher level than disruption of well-known BCL11A binding site in erythroblasts derived from human CD34+ hematopoietic stem and progenitor cells (HSPC).
View Article and Find Full Text PDFBeta-hemoglobinopathies are caused by mutations in the β-globin gene. One strategy to cure this disease relies on re-activating the γ-globin expression. BCL11A is an important transcription factor that suppresses the γ-globin expression, which makes it one of the most promising therapeutic targets in β-hemoglobinopathies.
View Article and Find Full Text PDFAnemia of inflammation, also known as anemia of chronic disease, is refractory to erythropoietin (EPO) treatment, but the mechanisms underlying the EPO refractory state are unclear. Here, we demonstrate that high mobility group box-1 protein (HMGB1), a damage-associated molecular pattern molecule recently implicated in anemia development during sepsis, leads to reduced expansion and increased death of EPO-sensitive erythroid precursors in human models of erythropoiesis. HMGB1 significantly attenuates EPO-mediated phosphorylation of the Janus kinase 2/STAT5 and mTOR signaling pathways.
View Article and Find Full Text PDFCongenital dyserythropoietic anemia type II (CDAII) results from loss-of-function mutations in . In contrast to humans, SEC23B-deficient mice deletion do not exhibit CDAII but die perinatally with pancreatic degeneration. Here, we demonstrate that expression of the full SEC23A protein (the SEC23B paralog) from the endogenous regulatory elements of completely rescues the SEC23B-deficient mouse phenotype.
View Article and Find Full Text PDFEx vivo manufactured red blood cells (RBC) generated from immortalized erythroid cell lines which can continuously grow are expected to become a significant alternative in future transfusion therapies. The ectopic expression of human papilloma virus (HPV) E6/E7 gene has successfully been employed to establish these cell lines. To induce differentiation and maturation of the immortalized cell lines, terminating the HPV-E6/E7 expression through a gene induction system has been believed to be essential.
View Article and Find Full Text PDFMutations in the adult β-globin gene can lead to a variety of hemoglobinopathies, including sickle cell disease and β-thalassemia. An increase in fetal hemoglobin expression throughout adulthood, a condition named hereditary persistence of fetal hemoglobin (HPFH), has been found to ameliorate hemoglobinopathies. Deletional HPFH occurs through the excision of a significant portion of the 3' end of the β-globin locus, including a CTCF binding site termed 3'HS1.
View Article and Find Full Text PDFDeveloping robust methodology for the sustainable production of red blood cells is essential for providing an alternative source of clinical-quality blood, particularly for individuals with rare blood group phenotypes. Immortalized erythroid progenitor cell lines are the most promising emergent technology for achieving this goal. We previously created the erythroid cell line BEL-A from bone marrow CD34 cells that had improved differentiation and enucleation potential compared to other lines reported.
View Article and Find Full Text PDFHuman fetal globin (γ-globin) genes are developmentally silenced after birth, and reactivation of γ-globin expression in adulthood ameliorates symptoms of hemoglobin disorders, such as sickle cell disease (SCD) and β-thalassemia. However, the mechanisms by which γ-globin expression is precisely regulated are still incompletely understood. Here, we found that NonO (non-POU domain-containing octamer-binding protein) interacted directly with SOX6, and repressed the expression of γ-globin gene in human erythroid cells.
View Article and Find Full Text PDFImmortalized erythroid cell lines are expected to be a promising source of ex vivo manufactured red blood cells (RBCs), however the induction of enucleation in these cell lines is inefficient at present. We utilized an imaging-based high-throughput system to identify chemical compounds that trigger enucleation of human erythroid cell lines. Among >3,300 compounds, we identified multiple histone deacetylase inhibitors (HDACi) inducing enucleated cells from the cell line, although an increase in membrane fragility of enucleated cells was observed.
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