Base-editing-mediated dissection of a γ-globin cis-regulatory element for the therapeutic reactivation of fetal hemoglobin expression

Antoniou, Panagiotis; Hardouin, Giulia; Martinucci, Pierre; Frati, Giacomo; Félix, Tristan; Chalumeau, Anne; Fontana, Letizia; Martin, J.; Masson, Cécile; Brusson, Mégane; Maule, Giulia; Rosello, Marion; Giovannangéli, Carine; Abramowski, Vincent; Villartay, Jean‐Pierre de; Concordet, Jean‐Paul; Bene, Filippo Del; Nemer, Wassim El; Amendola, Mario; Cavazzana, Marina; Cereseto, Anna; Romano, Oriana; Miccio, Annarita

doi:10.1038/s41467-022-34493-1

Cited by 39 publications

(23 citation statements)

References 77 publications

(102 reference statements)

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“…Importantly, Zeng et al , suggested that two rounds of electroporation were required to achieve high editing efficiencies by using the A3A (N57Q)-BE3 editor as RNP complex with a sgRNA targeting the +58 BCL11A erythroid enhancer ( Zeng et al, 2020 ). However, in this study and in a recent study by Antoniou et al , it is suggested that one mRNA-based nucleofection of BEs is adequate to reach high efficiencies in HSPCs ( Antoniou et al, 2022 ). Furthermore, deconvolution of sequence traces did not show any edits outside the predicted editing window (position 4–8 of sgRNA) for BCL11A and HBG as targets, and only the intended transition events (C>T) and no transversion events (C>A, C>G) were detected above background.…”

Section: Discussionmentioning

confidence: 46%

“…By contrast, mRNA- or mRNA/gRNA-based delivery offer near-universal applicability, high speed of adoption and application, low toxicity and high editing efficiencies. For the example of hemoglobinopathies, a growing body of work demonstrates that delivery of in vitro transcribed genome editing tools, such as BEs and TALENs in sickle-cell-disease and β-thalassemic cells, achieved high levels of functional correction and reached high editing efficiencies (40%–80%) ( Patsali et al, 2019b ; Gaudelli et al, 2020 ; Newby et al, 2021 ; Antoniou et al, 2022 ). Depending on the setup, mRNA-based delivery might vastly outperform editing efficiency achieved by RNPs for BEs ( Newby et al, 2021 ), while potentially modulating viral and immune transcriptional signatures and overall reducing DNA-damage, cell-cycle and metabolic response pathways in HSPCs ( Cromer et al, 2018 ).…”

Section: Discussionmentioning

confidence: 99%

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High-efficiency editing in hematopoietic stem cells and the HUDEP-2 cell line based on in vitro mRNA synthesis

Papaioannou

Patsali²,

Naiisseh³

et al. 2023

Front. Genome Ed.

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Introduction: 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 in vitro manipulation. Moreover, ex vivo 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.Methods: Here, we detail an in vitro 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 in vitro delivery of genome editing tools.Results and Discussion: 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.

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Section: Discussionmentioning

confidence: 46%

Section: Discussionmentioning

confidence: 99%

High-efficiency editing in hematopoietic stem cells and the HUDEP-2 cell line based on in vitro mRNA synthesis

Papaioannou

Patsali²,

Naiisseh³

et al. 2023

Front. Genome Ed.

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“…Human CD45 + cells were differentiated into mature RBCs using a three-phase erythroid differentiation protocol 51,52 .…”

Section: Ex Vivo Erythroid Differentiation Of Human Cd45 + Cellsmentioning

confidence: 99%

Safety and efficacy study of CRISPR/Cas9 treatment of sickle cell disease in clinically relevant conditions highlights disease-specific response

Frati,

Brusson,

Sartre

et al. 2024

Preprint

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Reactivation of fetal hemoglobin (HbF) expression through clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9-mediated disruption of regulatory elements involved in γ-globin gene repression is a promising gene therapy strategy for the treatment of sickle cell disease (SCD). However, preclinical studies aimed at optimizing the genome editing process and evaluating the safety of the editing strategy are necessary to translate this approach to the clinics. This is particularly relevant in the context of SCD, a disease characterized by inflammation, which can affect hematopoietic stem and progenitor cells (HSPCs), the target cell population in gene therapy approaches for hematopoietic disorders. Here, we describe a genome editing strategy leading to therapeutically relevant reactivation of HbF expression by targeting the binding sites (BSs) for the leukemia/lymphoma related factor (LRF) transcriptional repressor in the HBG1 and HBG2 γ-globin promoters. Electroporation of Cas9 ribonucleoprotein and single guide RNA (sgRNA) targeting the HBG promoters in healthy donor (HD) and patient-derived HSPCs resulted in a high frequency of LRF BS disruption and potent HbF synthesis in their erythroid progeny differentiated in vitro and ex vivo after transplantation into immunodeficient mice. LRF BS disruption did not impair SCD and HD HSPC engraftment and differentiation, but was more efficient in SCD than in HD cells. However, SCD HSPCs showed a reduced engraftment and a myeloid bias compared to HD cells. Importantly, in primary HSPCs, we detected off-target activity and the intra- and inter-chromosomal rearrangements between on- and off-target sites, which were more pronounced in SCD samples (likely because of the higher overall editing efficiency), but did not impact the target gene expression. Off-target activity was observed in vitro and in vivo, thus indicating that it does not impair engraftment and differentiation of both SCD and HD HSPCs. Finally, transcriptomic analyses showed that the genome editing procedure results in the upregulation of genes involved in DNA damage and inflammatory responses in both HD and SCD samples, although gene dysregulation was more evident in SCD HSPCs. Overall, this study provides evidences of feasibility, efficacy and safety for a genome editing strategy based on HbF reactivation and highlights the need of performing safety studies, when possible, in clinically relevant conditions, i.e., in patient-derived HSPCs.

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“…Because single-strand nicks are repaired by the high-fidelity base-excision repair pathway, BEs have been claimed to reduce on-target and off-target effects ( 36 ). However, recent data demonstrated deletion of a 4.9 kb region after base editing of the HBG1/HBG2 promoters, indicating that also base editing can induce structural variations ( 76 ). Furthermore, bystander editing effects ( 77 ) and gRNA-independent off-target activities on both DNA and RNA ( 78 , 79 ) have been described for both ABEs and CBEs.…”

Section: Off-target Effectsmentioning

confidence: 99%

Clinical genome editing to treat sickle cell disease—A brief update

Zarghamian

Klermund²,

Cathomen³

2023

Front. Med.

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Sickle cell disease (SCD) is one of the most common hemoglobinopathies. Due to its high prevalence, with about 20 million affected individuals worldwide, the development of novel effective treatments is highly warranted. While transplantation of allogeneic hematopoietic stem cells (HSC) is the standard curative treatment approach, a variety of gene transfer and genome editing strategies have demonstrated their potential to provide a prospective cure for SCD patients. Several stratagems employing CRISPR-Cas nucleases or base editors aim at reactivation of γ-globin expression to replace the faulty β-globin chain. The fetal hemoglobin (HbF), consisting of two α-globin and two γ-globin chains, can compensate for defective adult hemoglobin (HbA) and reverse the sickling of hemoglobin-S (HbS). Both disruption of cis-regulatory elements that are involved in inhibiting γ-globin expression, such as BCL11A or LRF binding sites in the γ-globin gene promoters (HBG1/2), or the lineage-specific disruption of BCL11A to reduce its expression in human erythroblasts, have been demonstrated to reestablish HbF expression. Alternatively, the point mutation in the HBB gene has been corrected using homology-directed repair (HDR)-based methodologies. In general, genome editing has shown promising results not only in preclinical animal models but also in clinical trials, both in terms of efficacy and safety. This review provides a brief update on the recent clinical advances in the genome editing space to offer cure for SCD patients, discusses open questions with regard to off-target effects induced by the employed genome editors, and gives an outlook of forthcoming developments.

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Base-editing-mediated dissection of a γ-globin cis-regulatory element for the therapeutic reactivation of fetal hemoglobin expression

Cited by 39 publications

References 77 publications

High-efficiency editing in hematopoietic stem cells and the HUDEP-2 cell line based on in vitro mRNA synthesis

High-efficiency editing in hematopoietic stem cells and the HUDEP-2 cell line based on in vitro mRNA synthesis

Safety and efficacy study of CRISPR/Cas9 treatment of sickle cell disease in clinically relevant conditions highlights disease-specific response

Clinical genome editing to treat sickle cell disease—A brief update

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