Correction of IVS I-110(G>A) β-thalassemia by CRISPR/Cas-and TALEN-mediated disruption of aberrant regulatory elements in human hematopoietic stem and progenitor cells β-Hemoglobinopathies result from mutations in the β-globin (HBB) gene. 1 Whereas causative mutations may be corrected by precise gene correction based on homology-directed repair, imprecise disruption of genome elements by non-homologous end joining is inherently more efficient and more suitable for long-term repopulating cells. 2 This has already prompted the pursuit of disruption-based reactivation of the HBB paralog g-globin as a potentially universal genome-editing strategy to treat patients with β-hemoglobinopathies, 3 which is as yet unproven in the clinic. The common β-thalassemia allele IVSI-110 (HBB ) has an aberrant splice acceptor site that leads to abnormal splicing. 4 Here we investigated the use of a mutation-specific and disruption-based approach to correct HBB . Based on both transcription activator-like effector nucleases (TALEN) and CRISPR/Cas9 RNA-guided HBB -targeting nucleases we analyzed non-homologous end joining-based indel events at on-and off-target sites, and the efficiency of functional correction in patient-derived CD34 + -derived HBB IVS-110(G>A) -homozygous erythroblasts. Both platforms showed significant correction at the RNA, protein and morphological levels, with up to 95% on-target disruption, using a design that minimized d-globin (HBD) offtarget activity. The present study establishes suitable target sequences for effective restoration of normal splicing and validates gene disruption by virus-and DNA-free delivery of nucleases as potential therapy for HBB thalassemia.The HBB mutation resides 19 nucleotides upstream of the normal intron-1 splice acceptor site. We identified one CRISPR/Cas9 and two TALEN-pair target sites compatible with platform-specific sequence constraints, proximity of exon 2, and the need to discern HBB from HBD for therapy by disruption ( Figure 1, Online Supplementary Figure S1). Predicted double-stranded break sites were adjacent to the aberrant splice acceptor site for the RNA-guided nuclease (RGN) and upstream for TALEN pairs, TALEN R1/L1 (R1/L1) and TALEN
The β-hemoglobinopathies sickle cell anemia and β-thalassemia are the focus of many gene-therapy studies. A key disease parameter is the abundance of globin chains because it indicates the level of anemia, likely toxicity of excess or aberrant globins, and therapeutic potential of induced or exogenous β-like globins. Reversed-phase high-performance liquid chromatography (HPLC) allows versatile and inexpensive globin quantification, but commonly applied protocols suffer from long run times, high sample requirements, or inability to separate murine from human β-globin chains. The latter point is problematic for in vivo studies with gene-addition vectors in murine disease models and mouse/human chimeras. This study demonstrates HPLC-based measurements of globin expression (1) after differentiation of the commonly applied human umbilical cord blood–derived erythroid progenitor-2 cell line, (2) in erythroid progeny of CD34+ cells for the analysis of clustered regularly interspaced short palindromic repeats/Cas9-mediated disruption of the globin regulator BCL11A, and (3) of transgenic mice holding the human β-globin locus. At run times of 8 min for separation of murine and human β-globin chains as well as of human γ-globin chains, and with routine measurement of globin-chain ratios for 12 nL of blood (tested for down to 0.75 nL) or of 300,000 in vitro differentiated cells, the methods presented here and any variant-specific adaptations thereof will greatly facilitate evaluation of novel therapy applications for β-hemoglobinopathies.
This study seeks to evaluate the impact of uncertainty in the pre-retrofit thermal performance of solid walls of English dwellings on post-retrofit energy use. Five dwelling archetypes, broadly representative of English solid wall properties, were modelled pre-and post-retrofit, under different wall insulation scenarios, using dynamic thermal simulation. Findings indicate that whilst solid wall insulation could result in a significant reduction of space heating demand, uncertainties in the preretrofit solid wall U-value could lead to a gap between the anticipated and actual energy performance. Specifically, results show that if the current U-value assumption of 2.1 W/m 2 K is indeed an overestimation of the in-situ U-value of solid walls, then the anticipated carbon savings could be significantly reduced by up to 65%. Practical Application:The performance gap observed in this study revealed that the actual carbon savings arising from the retrofit of solid wall properties could be significantly lower than predicted. This will not only affect UK Government carbon reduction targets, but it can also result in a lack of confidence amongst stakeholders who may consequently doubt the effectiveness of energy retrofit measures, thus reducing their uptake. Uncertainties regarding solid wall U-values may necessitate the reexamination of the carbon targets set for the retrofit of solid wall dwellings and the exploration of alternative ways to further reduce their carbon emissions, e.g. by specifying higher insulation thicknesses.2
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 BCL11A in erythroid cells by tagging the 3′ untranslated region (UTR) of BCL11A 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.
5-Azacitidine (5-AZA) is widely used for the treatment of higher-risk myelodysplastic syndromes. However, response and survival rates vary considerably, while indicated treatment duration remains undefined. For these reasons, factors determining response and survival are of major importance. Clinical, morphological, flow cytometry, cytogenetic and molecular factors are discussed in this review. Biomarkers predictive of response and prognosis, as well as their link to the mode of action of 5-AZA are also addressed, shifting the focus from clinical practice to investigational research. Their use could further improve prognostic classification of 5-AZA treated higher-risk myelodysplastic syndromes in the near future.
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