Short-hairpin RNA against aberrant HBB^{IVSI-110(G>A)} mRNA restores β-globin levels in a novel cell model and acts as mono- and combination therapy for β-thalassemia in primary hematopoietic stem cells

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

“…Likewise, hemoglobinization was increased with the RGN (90.7±4.1%, P=0.0688) and significantly increased with R1/L2 (91.0±5.1%, P=0.0272) compared with controls (85.6±3.3%) ( Figure 3C-E), although this represents a less sensitive indicator of functional correction for β-thalassemias with residual β-globin expression. 4 RNA analysis of variant ratios revealed significantly corrected HBB pre-mRNA splicing in bulk populations, from a control ratio of 56.8±0.9% to 92.0±3.7% (P<0.0001) for R1/L2 and to 99.3±1.3% (P<0.0001) for RGN treatment (Figure 3F, top), the latter with a real-time quantitative PCR readout for aberrant mRNA close to the limit of detection. As an additional measurement, total HBB mRNA indicated variably increased expression by a factor of 2.19±1.39 for R1/L2 and of 2.36±2.16 for RGN treatment (Figure 3F, bottom).…”

“…We analyzed treatment-related functional correction based on key disease parameters of HBBI thalassemia, specifically erythropoiesis and hemoglobinization by differential microscopic scoring, HBB mRNA splicing by real-time quantitative PCR and expression of individual globin species by RP-HPLC. 4,15 Microscopy consistently showed morphology indicative of more advanced erythroid differentiation after R1/L2 and RGN treatment ( Figure 3B), which based on stalling of thalassemic progenitors at the polychromatophilic stage of erythropoiesis is a diagnostic gold standard for disease correction, at moderate sample requirements. 4 Treatment-blinded scoring of thousands of cells from each culture (R1/L2: 4,579; RGN: 2,287; GFP: 4,230; untransfected control: 4,570) showed significant correction of late-stage erythroid differentiation for R1/L2 (64.8±6.9%, P<0.0001) and for the RGN (67.0±6.4%, P<0.0001) compared with controls (51.0±6.9) ( Figure 3C, D).…”

“…4,15 Microscopy consistently showed morphology indicative of more advanced erythroid differentiation after R1/L2 and RGN treatment ( Figure 3B), which based on stalling of thalassemic progenitors at the polychromatophilic stage of erythropoiesis is a diagnostic gold standard for disease correction, at moderate sample requirements. 4 Treatment-blinded scoring of thousands of cells from each culture (R1/L2: 4,579; RGN: 2,287; GFP: 4,230; untransfected control: 4,570) showed significant correction of late-stage erythroid differentiation for R1/L2 (64.8±6.9%, P<0.0001) and for the RGN (67.0±6.4%, P<0.0001) compared with controls (51.0±6.9) ( Figure 3C, D). Likewise, hemoglobinization was increased with the RGN (90.7±4.1%, P=0.0688) and significantly increased with R1/L2 (91.0±5.1%, P=0.0272) compared with controls (85.6±3.3%) ( Figure 3C-E), although this represents a less sensitive indicator of functional correction for β-thalassemias with residual β-globin expression.…”

HbS/β+ thalassemia: Really a mild disease? A National survey from the AIEOP Sickle Cell Disease Study Group with genotype‐phenotype correlation

“…Some promoter mutations are ethnic‐specific; −29 A > G and −88 C > T are frequent in Africa while −87 C > T and −101 C > T are more common in Mediterranean areas . Among the β+ mutations, the IVS‐I‐110 is one of the commonest β+ thalassemia allele in the Mediterranean area; it is due to an aberrant splice acceptor site that leads to abnormal splicing, thus creating an aberrant mRNA containing a 19‐nt intronic fragment with an in‐frame stop codon . This mutation is associated with a variable amount of HbA …”

“…8 Among the β+ mutations, the IVS-I-110 is one of the commonest β+ thalassemia allele in the Mediterranean area 9 ; it is due to an aberrant splice acceptor site that leads to abnormal splicing, thus creating an aberrant mRNA containing a 19-nt intronic fragment with an in-frame stop codon. 10 This mutation is associated with a variable amount of HbA. 11 While a large amount of data are available in the literature concerning the diagnosis and care of the severe SCD phenotypes, management of S/β+ patients is still a matter of debate, because of the rarity of this condition and the limited phenotype available data, [11][12][13][14][15][16] with the exception of a large study restricted to the Jamaican population.…”