Initiation of regular transfusion in transfusion-dependent thalassemia (TDT) is based on the assessment of clinical phenotype. Pathogenic HBB variants causing β-thalassemia are important determinants ...of phenotype and could be used to aid decision making. We investigated the association of HBB genotype with survival in a cohort study in the four thalassemia centres in Cyprus. HBB genotype was classified as severe (β0/β0 or β+/β0), moderate (β+/β+), or mild (β0/β++ or β+/β++). Risk factors for mortality were evaluated using multivariate Cox proportional-hazards regression. 537 subjects were followed for a total of 20,963 person years. 80.4% (95% CI 76.4-84.7) of individuals survived to 50 years of age with increasing rates of liver, infection and malignancy-related deaths observed during recent follow-up. We evaluated non-modifiable risk factors and found worse outcomes associated with male sex (Hazard ratio 1.9, 95% CI 1.1-3.0, p=0.01) and milder genotype (Hazard ratio 1.6, 95% CI 1.1-2.3, p=0.02). The effect of genotype was confirmed in a second model, which included treatment effects. Patients with a milder genotype initiated transfusion significantly later and had reduced blood requirements compared to those with moderate or severe genotypes, although pre-transfusion hemoglobin levels did not differ between genotypes. Our results suggest that early treatment decisions to delay transfusion and different long-term treatment strategies in milder genotypes have led to adverse long-term effects of under-treated thalassemia and worse survival. We propose that HBB genotype determination and use of this information to aid in decision making can improve long-term outcomes of thalassaemia patients.
MicroRNAs (miRNAs) are small non-coding RNAs crucial for post-transcriptional and translational regulation of cellular and developmental pathways. The study of miRNAs in erythropoiesis elucidates ...underlying regulatory mechanisms and facilitates related diagnostic and therapy development. Here, we used DNA Nanoball (DNB) small RNA sequencing to comprehensively characterize miRNAs in human erythroid cell cultures. Based on primary human peripheral-blood-derived CD34+ (hCD34+) cells and two influential erythroid cell lines with adult and fetal hemoglobin expression patterns, HUDEP-2 and HUDEP-1, respectively, our study links differential miRNA expression to erythroid differentiation, cell type, and hemoglobin expression profile. Sequencing results validated by reverse-transcription quantitative PCR (RT-qPCR) of selected miRNAs indicate shared differentiation signatures in primary and immortalized cells, characterized by reduced overall miRNA expression and reciprocal expression increases for individual lineage-specific miRNAs in late-stage erythropoiesis. Despite the high similarity of same-stage hCD34+ and HUDEP-2 cells, differential expression of several miRNAs highlighted informative discrepancies between both cell types. Moreover, a comparison between HUDEP-2 and HUDEP-1 cells displayed changes in miRNAs, transcription factors (TFs), target genes, and pathways associated with globin switching. In resulting TF-miRNA co-regulatory networks, major therapeutically relevant regulators of globin expression were targeted by many co-expressed miRNAs, outlining intricate combinatorial miRNA regulation of globin expression in erythroid cells.
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IZUM, KILJ, NUK, PILJ, PNG, SAZU, UL, UM, UPUK
The β-thalassemias are an increasing challenge to health systems worldwide, caused by absent or reduced β-globin (HBB) production. Of particular frequency in many Western countries is HBBIVSI−110(G > ...A) β-thalassemia (HGVS name: HBB:c.93-21G > A). Its underlying mutation creates an abnormal splice acceptor site in the HBB gene, and while partially retaining normal splicing of HBB, it severely reduces HBB protein expression from the mutant locus and HBB loci in trans. For the assessment of the underlying mechanisms and of therapies targeting β-thalassemia, accurate quantification of aberrant and normal HBB mRNA is essential, but to date, has only been performed by approximate methods. To address this shortcoming, we have developed an accurate, duplex reverse-transcription quantitative PCR assay for the assessment of the ratio and absolute quantities of normal and aberrant mRNA species as a tool for basic and translational research of HBBIVSI−110(G > A) β-thalassemia. The method was employed here to determine mRNA ratios and quantities in blood and primary cell culture samples and correlate them with HBB protein levels. Moreover, with its immediate utility for β-thalassemia and the mutation in hand, the approach can readily be adopted for analysis of alternative splicing or for quantitative assays of any disease-causing mutation that interferes with normal splicing.
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IZUM, KILJ, NUK, PILJ, PNG, SAZU, UL, UM, UPUK
Therapy via the gene addition of the anti-sickling βAS3-globin transgene is potentially curative for all β-hemoglobinopathies and therefore of particular clinical and commercial interest. This study ...investigates GLOBE-based lentiviral vectors (LVs) for βAS3-globin addition and evaluates strategies for an increased β-like globin expression without vector dose escalation. First, we report the development of a GLOBE-derived LV, GLV2-βAS3, which, compared to its parental vector, adds anti-sickling action and a transcription-enhancing 848-bp transcription terminator element, retains high vector titers and allows for superior β-like globin expression in primary patient-derived hematopoietic stem and progenitor cells (HSPCs). Second, prompted by our previous correction of HBBIVSI−110(G>A) thalassemia based on RNApol(III)-driven shRNAs in mono- and combination therapy, we analyzed a series of novel LVs for the RNApol(II)-driven constitutive or late-erythroid expression of HBBIVSI−110(G>A)-specific miRNA30-embedded shRNAs (shRNAmiR). This included bifunctional LVs, allowing for concurrent βAS3-globin expression. LVs were initially compared for their ability to achieve high β-like globin expression in HBBIVSI−110(G>A)-transgenic cells, before the evaluation of shortlisted candidate LVs in HBBIVSI−110(G>A)-homozygous HSPCs. The latter revealed that β-globin promoter-driven designs for monotherapy with HBBIVSI−110(G>A)-specific shRNAmiRs only marginally increased β-globin levels compared to untransduced cells, whereas bifunctional LVs combining miR30-shRNA with βAS3-globin expression showed disease correction similar to that achieved by the parental GLV2-βAS3 vector. Our results establish the feasibility of high titers for LVs containing the full HBB transcription terminator, emphasize the importance of the HBB terminator for the high-level expression of HBB-like transgenes, qualify the therapeutic utility of late-erythroid HBBIVSI−110(G>A)-specific miR30-shRNA expression and highlight the exceptional potential of GLV2-βAS3 for the treatment of severe β-hemoglobinopathies.
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IZUM, KILJ, NUK, PILJ, PNG, SAZU, UL, UM, UPUK
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. Moreover,
editing of CD34
cells has immediate therapeutic relevance. Both cell types are sensitive to standard transfection procedures and reagents, such as lipofection with plasmid DNA, calling for more suitable methodology in order to achieve high efficiency and tolerability of editing with editors of choice. These challenges can be addressed by RNA delivery, either as a mixture of guide RNA and mRNA for CRISRP/Cas-based systems or as a mixture of mRNAs for TALENs. Compared to ribonucleoproteins or proteins, RNA as vector creates flexibility by removing dependence on commercial availability or laborious in-house preparations of novel editor proteins. Compared to DNA, RNA is less toxic and by obviating nuclear transcription and export of mRNA offers faster kinetics and higher editing efficiencies.
Here, we detail an
transcription protocol based on plasmid DNA templates with the addition of Anti-Reverse Cap Analog (ARCA) using T7 RNA polymerase, and poly (A) tailing using poly (A) polymerase, combined with nucleofection of HUDEP-2 and patient-derived CD34
cells. Our protocol for RNA-based delivery employs widely available reagents and equipment and can easily be adopted for universal
delivery of genome editing tools.
Drawing on a common use case, we employ the protocol to target a β-globin mutation and to reactivate γ-globin expression as two potential therapies for β-hemoglobinopathies, followed by erythroid differentiation and functional analyses. Our protocol allows high editing efficiencies and unimpaired cell viability and differentiation, with scalability, suitability for functional assessment of editing outcomes and high flexibility in the application to different editors.
Reactivation of γ-globin is considered a promising approach for the treatment of β-thalassemia and sickle cell disease. Therapeutic induction of γ-globin expression, however, is fraught with lack of ...suitable therapeutic targets. The aim of this study was to investigate the effects that treatment with decitabine has on the proteome of human primary erythroid cells from healthy and thalassemic volunteers, as a means of identifying new potential pharmacological targets. Decitabine is a known γ-globin inducer, which is not, however, safe enough for clinical use. A proteomic approach utilizing isobaric tags for relative and absolute quantitation (iTRAQ) analysis, in combination with high-pH reverse phase peptide fractionation followed by liquid chromatography-tandem mass spectrometry (LC-MS/MS), was employed to investigate the effects of decitabine treatment. Bioinformatics analysis making use of the Database for Annotation, Visualization and Integrated Discovery (DAVID) was employed for functional annotation of the 192 differentially expressed proteins identified. The data are available via ProteomeXchange with identifier PXD006889. The proteins fall into various biological pathways, such as the NF-κB signaling pathway, and into many functional categories including regulation of cell proliferation, transcription factor and DNA binding, protein stabilization, chromatin modification and organization, and oxidative stress proteins.
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