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.
Molecular 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. To this end, we employed nucleofection of CRISPR/Cas9 ribonucleoprotein (RNP) particles alongside double- or single-stranded oligodeoxynucleotides for, respectively, non-homologous-end-joining (NHEJ)- or homology-directed-repair (HDR)-mediated MRS insertion. NHEJ-based tagging was imprecise and inefficient (≤6%) and uniformly produced knock-in- and indel-containing MRS tags, whereas HDR-based tagging was more efficient (≤18%), but toxic for longer donors encoding concatenated and thus potentially more efficient MRS tags. Isolation of clones for robust HEK293T cells tagged with a homozygous quadruple MRS resulted in 25% spontaneous reduction in BCL11A and up to 36% reduction after transfection with an miR-451a mimic. Isolation of clones for human umbilical cord blood-derived erythroid progenitor-2 (HUDEP-2) cells tagged with single or double MRS allowed detection of albeit weak γ-globin induction. Our study demonstrates suitability of TAMED for physiologically relevant modulation of gene expression and its unsuitability for therapeutic application in its current form.
β-Thalassemia is brought about by defective β-globin (HBB hemoglobin subunit β) formation and, in severe cases, requires regular blood transfusion and iron chelation for survival. Genome editing of ...hematopoietic stem cells allows correction of underlying mutations as curative therapy. As potentially safer alternatives to double-strand-break–based editors, base editors (BEs) catalyze base transitions for precision editing of DNA target sites, prompting us to reclone and evaluate two recently published adenine BEs (ABEs; SpRY and SpG) with relaxed protospacer adjacent motif requirements for their ability to correct the common HBBIVSI-110(G>A) splice mutation. Nucleofection of ABE components as RNA into patient-derived CD34+ cells achieved up to 90% editing of upstream sequence elements critical for aberrant splicing, allowing full characterization of the on-target base-editing profile of each ABE and the detection of differences in on-target insertions and deletions. In addition, this study identifies opposing effects on splice correction for two neighboring context bases, establishes the frequency distribution of multiple BE editing events in the editing window, and shows high-efficiency functional correction of HBBIVSI-110(G>A) for our ABEs, including at the levels of RNA, protein, and erythroid differentiation.
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Lederer and colleagues have targeted HBBIVSI-110(G>A) thalassemia with novel base editors and shown that editing of an upstream base results in phenotypic correction. Alternatively, combinatorial base editing of three HBBIVSI-110(G>A)-proximal upstream bases has similarly high correction efficiency. By contrast, editing of an interspersed base is instead detrimental to normal splicing.
Advanced therapy medicinal products (ATMPs) are medicines for human use based on genes, cells or tissue engineering. After clear successes in adults, the nascent technology now sees increasing ...pediatric application. For many still untreatable disorders with pre- or perinatal onset, timely intervention is simply indispensable; thus, prenatal and pediatric applications of ATMPs hold great promise for curative treatments. Moreover, for most inherited disorders, early ATMP application may substantially improve efficiency, economy and accessibility compared with application in adults. Vindicating this notion, initial data for cell-based ATMPs show better cell yields, success rates and corrections of disease parameters for younger patients, in addition to reduced overall cell and vector requirements, illustrating that early application may resolve key obstacles to the widespread application of ATMPs for inherited disorders. Here, we provide a selective review of the latest ATMP developments for prenatal, perinatal and pediatric use, with special emphasis on its comparison with ATMPs for adults. Taken together, we provide a perspective on the enormous potential and key framework parameters of clinical prenatal and pediatric ATMP application.
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.
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.
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.
Haemoglobinopathies are common monogenic disorders with diverse clinical manifestations, partly attributed to the influence of modifier genes. Recent years have seen enormous growth in the amount of ...genetic data, instigating the need for ranking methods to identify candidate genes with strong modifying effects. Here, we present the first evidence-based gene ranking metric (IthaScore) for haemoglobinopathy-specific phenotypes by utilising curated data in the IthaGenes database. IthaScore successfully reflects current knowledge for well-established disease modifiers, while it can be dynamically updated with emerging evidence. Protein-protein interaction (PPI) network analysis and functional enrichment analysis were employed to identify new potential disease modifiers and to evaluate the biological profiles of selected phenotypes. The most relevant gene ontology (GO) and pathway gene annotations for (a) haemoglobin (Hb) F levels/Hb F response to hydroxyurea included urea cycle, arginine metabolism and vascular endothelial growth factor receptor (VEGFR) signalling, (b) response to iron chelators included xenobiotic metabolism and glucuronidation, and (c) stroke included cytokine signalling and inflammatory reactions. Our findings demonstrate the capacity of IthaGenes, together with dynamic gene ranking, to expand knowledge on the genetic and molecular basis of phenotypic variation in haemoglobinopathies and to identify additional candidate genes to potentially inform and improve diagnosis, prognosis and therapeutic management.
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