Forced chromatin looping raises fetal hemoglobin in adult sickle cells to higher levels than pharmacologic inducers

Breda, Laura; Motta, Irene; Lourenço, Sílvia Pires; Gemmo, Chiara; Deng, Wulan; Rupon, Jeremy; Abdulmalik, Osheiza; Manwani, Deepa; Blobel, Gerd A.; Rivella, Stefano

doi:10.1182/blood-2016-01-691089

Cited by 69 publications

(70 citation statements)

References 25 publications

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“…Thus, forced chromatin looping can override the g-to-b globin gene switch. 2,3 Synthetic zinc finger DNA-binding domains (ZF-DBDs) have also been used in genome editing and transcription modulation experiments; in particular, ZF-DBDs targeted to a known site associated with hereditary persistence of fetal hemoglobin (HPFH), a benign genetic condition in which mutations in a sequence located at 567 bp upstream of the g-globin promoter attenuate g-to-b switching. The ZF-DBD that interacted with the targeted upstream sequence of g-globing promoter with high affinity increased the HbF level.…”

Section: Introductionmentioning

confidence: 99%

High level of fetal-globin reactivation by designed transcriptional activator-like effector

et al. 2020

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Key Points• TALEs targeted to the g-globin gene promoters reactivated their mRNA expression more than 70-fold with a collateral reduction in b-globin mRNA.• At day 19 of CD34 erythroid differentiation, TALEs increased g-globin more than 40fold in mRNA level and up to 70% of the total globin protein.The fetal-to-adult hemoglobin switch has been a focus of a long-standing effort to potentially treat sickle cell disease and b thalassemia by induction of fetal hemoglobin. In a continuation of this effort, we designed specific transcriptional activator-like effectors (TALEs) to target both the G g and A g-globin promoters. We fused the TALEs to a LIM domain binding protein (Ldb1) dimerization domain, followed by a T2A green fluorescent protein (GFP) cassette, which were assembled into a lentiviral vector. To prevent deletions caused by the repeats of TALEs during the lentivirus packing process, we changed the TALE encoding DNA by codon optimization. Intriguingly, 5 of 14 TALEs showed forced reactivation of fetalglobin expression in human umbilical cord blood-derived erythroid progenitor (HUDEP-2) cells, with a significant increase in the g-globin mRNA level by more than 70-fold. We also observed a more than 50% reduction of b-globin mRNA. High-performance liquid chromatography analysis revealed more than 30% fetal globin in TALE-induced cells compared with the control of 2%. Among several promoters studied, the b-globin gene promoter with the locus control region (LCR) enhancer showed the highest TALE expression during CD34 erythroid differentiation. At day 19 of differentiation, 2 TALEs increased fetalglobin expression more than 40-fold in the mRNA level and up to 70% of the total globin protein. These TALEs have potential for clinical translation.

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Section: Introductionmentioning

confidence: 99%

High level of fetal-globin reactivation by designed transcriptional activator-like effector

et al. 2020

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“…Apart from β- or γ-globin gene addition in hematopoietic stem cells by traditional lentiviral vectors, several other protocols aiming to increase or reactivate fetal hemoglobin (HbF) have been explored because this outcome would be applicable for amelioration of all β-globin disorders, irrespective of the genetic mutation. Tested strategies for HbF reactivation involve either forced chromatin looping, mediated by a lentiviral vector expressing the looping factor LDB1 linked to a zinc-finger protein binding the γ-globin promoter, 5 the re-creation of γ-globin promoter mutations leading to hereditary persistence of HbF (HPFH) conditions,6, 7 or downregulation or inhibition of trans -factors involved in γ-globin silencing 8 or disruption of their binding regions 9, 10, 11. Attempts to faithfully recreate HPFH γ-globin promoter mutations through gene editing have been associated with low efficiency in normal adult CD34+ cells ex vivo because less efficient homology- or microhomology-mediated repair is required 6, 7.…”

Section: Introductionmentioning

confidence: 99%

Disruption of the BCL11A Erythroid Enhancer Reactivates Fetal Hemoglobin in Erythroid Cells of Patients with β-Thalassemia Major

Psatha

Reik

Phelps

et al. 2018

Molecular Therapy - Methods & Clinical Development

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In the present report, we carried out clinical-scale editing in adult mobilized CD34+ hematopoietic stem and progenitor cells (HSPCs) using zinc-finger nuclease-mediated disruption of BCL11a to upregulate the expression of γ-globin (fetal hemoglobin). In these cells, disruption of the erythroid-specific enhancer of the BCL11A gene increased endogenous γ-globin expression to levels that reached or exceeded those observed following knockout of the BCL11A coding region without negatively affecting survival or in vivo long-term proliferation of edited HSPCs and other lineages. In addition, BCL11A enhancer modification in mobilized CD34+ cells from patients with β-thalassemia major resulted in a readily detectable γ-globin increase with a preferential increase in G-gamma, leading to an improved phenotype and, likely, a survival advantage for maturing erythroid cells after editing. Furthermore, we documented that both normal and β-thalassemia HSPCs not only can be efficiently expanded ex vivo after editing but can also be successfully edited post-expansion, resulting in enhanced early in vivo engraftment compared with unexpanded cells. Overall, this work highlights a novel and effective treatment strategy for correcting the β-thalassemia phenotype by genome editing.

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“…The characterization of BM‐derived HSPCs from patients with SCD is required to maximize the likelihood of success of gene therapy protocols for these patients, particularly as promising investigations are occurring regarding HSPC purification and gene transfer efficiency (Masiuk et al , ; Zonari et al , ), editing strategies (Breda et al , ; Dever et al , ; DeWitt et al , ; Hoban et al , ; Ye et al , ; Uchida et al , 2017a) and transplant conditioning (Chhabra et al , ) that relies on high quality HSPCs for success. We therefore sought to characterize the immunophenotype of CD34 + HSPCs from subjects with SCD (SCD BM) compared to that of healthy volunteers (non‐SCD BM) in addition to characterizing the inflammatory microenvironment in the BM within the MN layer.…”

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confidence: 99%

Bone marrow characterization in sickle cell disease: inflammation and stress erythropoiesis lead to suboptimal CD34 recovery

et al. 2019

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Summary Stress erythropoiesis and chronic inflammation in subjects with sickle cell disease (SCD) may have an impact on the bone marrow (BM) haematopoietic stem and progenitor cell (HSPC) quality and yield necessary for effective autologous, ex vivo HSPC gene therapy. BM from 19 subjects with SCD and five volunteers without SCD (non‐SCD) was collected in different anticoagulants and processed immediately (day 0) or the following day (day 1). Inflammatory, contamination and aggregation markers within the mononuclear layer, and CD34, CD45 and Glycophorin‐A (GPA) expression on HSPCs after CD34+ selection were analysed by conventional and imaging flow cytometry. Compared to non‐SCD BM, multiple markers of inflammation, contamination (red cells, P < 0·01; platelets, P < 0·01) and aggregates (platelet/granulocytes, P < 0·01; mononuclear/red cells, P < 0·01) were higher in SCD BM. Total CD34+ cell count was lower in SCD BM (P < 0·05), however CD34+ count was higher in SCD BM when collected in acid citrate dextrose‐A (ACDA) versus heparin (P < 0·05). Greater than 50% of CD34+ HSPCs from SCD BM are CD34dim due to higher erythroid lineage expression (P < 0·01) as single cell CD34+CD45+GPA+ (P < 0·01) and CD34+CD45−GPA+ (P < 0·01) HSPCs. SCD BM is characterized by increased inflammation, aggregation and contamination contributing to significant differences in HSPC quality and yield compared to non‐SCD BM.

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Forced chromatin looping raises fetal hemoglobin in adult sickle cells to higher levels than pharmacologic inducers

Abstract: Key Points Ldb1 transcription factor self-association domain fused to γ-globin promoter-specific ZF protein increases HbF, reduces HbS in hSCD cells. In vitro reactivation of HbF mediated by ZF-Ldb1 exceeds pharmacologic treatment in adult hSCD cells.

Cited by 69 publications

References 25 publications

High level of fetal-globin reactivation by designed transcriptional activator-like effector

High level of fetal-globin reactivation by designed transcriptional activator-like effector

Disruption of the BCL11A Erythroid Enhancer Reactivates Fetal Hemoglobin in Erythroid Cells of Patients with β-Thalassemia Major

Bone marrow characterization in sickle cell disease: inflammation and stress erythropoiesis lead to suboptimal CD34 recovery

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