Editing a γ-globin repressor binding site restores fetal hemoglobin synthesis and corrects the sickle cell disease phenotype

Weber, Leslie; Frati, Giacomo; Félix, Tristan; Hardouin, Giulia; Casini, Antonio; Wollenschlaeger, Clara; Meneghini, Vasco; Masson, Cécile; Cian, Anne De; Chalumeau, Anne; Mavilio, Fulvio; Amendola, Mario; André-Schmutz, Isabelle; Cereseto, Anna; Nemer, Wassim El; Concordet, Jean-Paul; Giovannangéli, Carine; Cavazzana, Marina; Miccio, Annarita

doi:10.1126/sciadv.aay9392

Cited by 97 publications

(109 citation statements)

References 54 publications

Supporting

Mentioning

107

Contrasting

Order By: Relevance

“…introduced three different HPFH deletions (3·5 Kb, 7·2 Kb Corfu region and 13·6 Kb, similar to the 12·9 Kb HPFH‐5 deletion). Weber et al ., 18 similarly to us, identified the ZBTB7A binding site as a potent target for the treatment of SCD.…”

Section: Discussionmentioning

confidence: 61%

Induction of therapeutic levels of HbF in genome‐edited primary β⁰39‐thalassaemia haematopoietic stem and progenitor cells

Mingoia

Caria

et al. 2020

Br J Haematol

View full text Add to dashboard Cite

Hereditary persistence of fetal haemoglobin (HPFH) is the major modifier of the clinical severity of b-thalassaemia. The homozygous mutation c.-196 C>T in the Ac-globin (HBG1) promoter, which causes Sardinian db 0-thalassaemia, is able to completely rescue the b-major thalassaemia phenotype caused by the b 0 39-thalassaemia mutation, ensuring high levels of fetal haemoglobin synthesis during adulthood. Here, we describe a CRISPR/Cas9 genome-editing approach, combined with the non-homologous end joining (NHEJ) pathway repair, aimed at reproducing the effects of this naturally occurring HPFH mutation in both HBG promoters. After selecting the most efficient guide RNA in K562 cells, we edited the HBG promoters in human umbilical cord blood-derived erythroid progenitor 2 cells (HUDEP-2) and in haematopoietic stem and progenitor cells (HSPCs) from b 0-thalassaemia patients to assess the therapeutic potential of HbF induction. Our results indicate that small deletions targeting the À196-promoter region restore high levels of fetal haemoglobin (HbF) synthesis in all cell types tested. In pools of HSPCs derived from homozygous b 0 39-thalassaemia patients, a 20% editing determined a parallel 20% increase of HbF compared to unedited pools. These results suggest that editing the region of HBG promoters around the À196 position has the potential to induce therapeutic levels of HbF in patients with most types of b-thalassaemia irrespective of the b-globin gene (HBB) mutations.

show abstract

Section: Discussionmentioning

confidence: 61%

Induction of therapeutic levels of HbF in genome‐edited primary β⁰39‐thalassaemia haematopoietic stem and progenitor cells

Mingoia

Caria

et al. 2020

Br J Haematol

View full text Add to dashboard Cite

show abstract

“…Consistently, the CRISPR-mediated disruption of these two binding sites resulted in a relevant increase in γ-globin expression (Traxler et al, 2016;Weber et al, 2020). Of note, the editing of the −158 ("XmnI-Gγ-site"), known to be influenced by a QTL on chromosome 8 (Garner et al, 2002), only marginally increased γ-globin expression (Weber et al, 2020), suggesting that possible background effects might be taken into account when considering editing for therapeutic purposes.…”

Section: The Editing Of Globin Genes Inspired By Hpfhmentioning

confidence: 76%

“…Thus, the editing of these γ-globin regions can result either in the mutation of a single or both HBG genes or in the deletion of the intergenic region, with different resulting percentages of γ-globin induction. Moreover, given the presence of short repeats within the promoter, MMEJ can also occur (Traxler et al, 2016;Weber et al, 2020). NHEJ can also be used to destroy the specific erythroid expression of repressors, such as BCL11A or, in principle, of LRF (both proteins have important roles in other hematopoietic cell types that must be preserved).…”

Section: The Editing Of Globin Genes Inspired By Hpfhmentioning

confidence: 99%

β-Hemoglobinopathies: The Test Bench for Genome Editing-Based Therapeutic Strategies

Barbarani

Labedz

2020

Front. Genome Ed.

View full text Add to dashboard Cite

Hemoglobin is a tetrameric protein composed of two α and two β chains, each containing a heme group that reversibly binds oxygen. The composition of hemoglobin changes during development in order to fulfill the need of the growing organism, stably maintaining a balanced production of α-like and β-like chains in a 1:1 ratio. Adult hemoglobin (HbA) is composed of two α and two β subunits (α2β2 tetramer), whereas fetal hemoglobin (HbF) is composed of two γ and two α subunits (α2γ2 tetramer). Qualitative or quantitative defects in β-globin production cause two of the most common monogenic-inherited disorders: β-thalassemia and sickle cell disease. The high frequency of these diseases and the relative accessibility of hematopoietic stem cells make them an ideal candidate for therapeutic interventions based on genome editing. These strategies move in two directions: the correction of the disease-causing mutation and the reactivation of the expression of HbF in adult cells, in the attempt to recreate the effect of hereditary persistence of fetal hemoglobin (HPFH) natural mutations, which mitigate the severity of β-hemoglobinopathies. Both lines of research rely on the knowledge gained so far on the regulatory mechanisms controlling the differential expression of globin genes during development.

show abstract

“…Accordingly, we suggest that for NHEJ-dependent strategies for therapeutic editing of HSCs, it may be beneficial to maintain HSC quiescence. Some CD34+ HSPC editing protocols appear to favor quiescent or G1 HSCs, whereas other protocols cause a higher frequency of editing in cycling or G2 HSCs (7, 11, 65, 68–70). Additionally, we suggest that for NHEJ applications, fusion of Cas9 to a G1-specific Cdt1 segment could be employed to restrict editing to G1 cells (68, 71), thereby minimizing the probability of micronucleus formation, chromosome segment missegregation during mitosis, and chromothripsis.…”

Section: Discussionmentioning

confidence: 99%

Chromothripsis as an on-target consequence of CRISPR-Cas9 genome editing

Leibowitz

Papathanasiou

Doerfler

et al. 2020

Preprint

View full text Add to dashboard Cite

Genome editing has promising therapeutic potential for genetic diseases and cancer (1, 2). However, the most practicable current approaches rely on the generation of DNA double-strand breaks (DSBs), which can give rise to a poorly characterized spectrum of structural chromosomal abnormalities. Here, we show that a catastrophic mutational process called chromothripsis is a previously unappreciated consequence of CRISPR-Cas9-mediated DSBs. Chromothripsis is extensive chromosome rearrangement restricted to one or a few chromosomes that can cause human congenital disease and cancer (3–6). Using model cell systems and a genome editing protocol similar to ones in clinical trials (7) (NCT03655678, NCT03745287) we show that CRISPR-Cas9-mediated DNA breaks generate abnormal nuclear structures—micronuclei and chromosome bridges—that trigger chromothripsis. Chromothripsis is an on-target toxicity that may be minimized by cell manipulation protocols or screening but cannot be completely avoided in many genome editing applications.

show abstract

Editing a γ-globin repressor binding site restores fetal hemoglobin synthesis and corrects the sickle cell disease phenotype

Cited by 97 publications

References 54 publications

Induction of therapeutic levels of HbF in genome‐edited primary β⁰39‐thalassaemia haematopoietic stem and progenitor cells

Induction of therapeutic levels of HbF in genome‐edited primary β⁰39‐thalassaemia haematopoietic stem and progenitor cells

β-Hemoglobinopathies: The Test Bench for Genome Editing-Based Therapeutic Strategies

Chromothripsis as an on-target consequence of CRISPR-Cas9 genome editing

Contact Info

Product

Resources

About

Editing a γ-globin repressor binding site restores fetal hemoglobin synthesis and corrects the sickle cell disease phenotype

Cited by 97 publications

References 54 publications

Induction of therapeutic levels of HbF in genome‐edited primary β039‐thalassaemia haematopoietic stem and progenitor cells

Induction of therapeutic levels of HbF in genome‐edited primary β039‐thalassaemia haematopoietic stem and progenitor cells

β-Hemoglobinopathies: The Test Bench for Genome Editing-Based Therapeutic Strategies

Chromothripsis as an on-target consequence of CRISPR-Cas9 genome editing

Contact Info

Product

Resources

About

Induction of therapeutic levels of HbF in genome‐edited primary β⁰39‐thalassaemia haematopoietic stem and progenitor cells

Induction of therapeutic levels of HbF in genome‐edited primary β⁰39‐thalassaemia haematopoietic stem and progenitor cells