Conversion of HbS to Hb G-Makassar By Adenine Base Editing Is Compatible with Normal Hemoglobin Function

Chu, Shih–Hsi; Ortega, Manuel; Feliciano, Paty; Winton, Valerie J.; Xu, Chavonna; Haupt, Daniel; McDonald, T. R. R.; Martinez, Salette; Liquori, Alexander; Marshall, Jeffrey; Lam, Daisy; Olins, Jenny; Rinaldi, Conrad; Rehberger, Kyle; Lazarra, Colin; Decker, Jeremy; Gantzer, Bob; Bohnuud, Tanggis; Born, David A.; Barrera, Luis; Yan, Bo; Slaymaker, Ian M.; Packer, Michael S.; Smith, Sarah; Zambonelli, C.; Lee, Matt; Gaudelli, Nicole M.; Hartigan, Adam J.; Ciaramella, Giuseppe

doi:10.1182/blood-2021-150922

Cited by 15 publications

(10 citation statements)

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“…Our cohort of cases is larger than the reported cases in Thailand The polymerisation of Hb is responsible for causing haemolysis, vasoocclusive crises and multi-organ damage in Hb S [17][18][19][20]. The approach on changing the Hb S to Hb G-Makassar is potentially encouraging to treat patients with SCD that could be explored in future clinical trials [21,22].…”

Section: Discussionmentioning

confidence: 99%

Clinical and haematological characteristics of 38 individuals with Hb G‐Makassar in Malaysia

Esa,

Mohamad,

Hamzah

et al. 2023

eJHaem

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Haemoglobin (Hb) G‐Makassar is a rare Hb variant. It presents a diagnostic challenge as it imitates sickle Hb (Hb S) in standard electrophoresis and high‐performance liquid chromatography assays requiring DNA analysis to confirm diagnosis. Both have point mutations in codon 6, exon 1 in the β‐globin (HBB) gene with different pathogenicities. This study describes the clinical phenotype, haematology and genotype of Hb G‐Makassar. Clinical and laboratory data of 38 cases of Hb G‐Makassar over 8 years were analysed. Hb G‐Makassar was confirmed by a direct sequencing of HBB gene and co‐inheritance of α‐thalassaemia determined through multiplex gap‐PCR and multiplex Amplification Refractory Mutation System polymerase chain reaction. All cases were Malays, predominantly from Terengganu (n = 20, 52.6%). There were 14 (36.8%) males and 24 (63.2%) females with median age of 25 years. Majority (n = 33, 86.8%) had features of thalassaemia trait with mean ± SD for Hb, mean cell volume (MCV) and mean cell haemoglobin (MCH) as 13.21 g/dL ± 1.69, 73.06 ± 4.48 fL and 24.71 ± 1.82 pg, respectively. None had evidence of haemolysis or thromboembolic complications. Six genotypes were identified; ßG‐Makassar/ß,αα/αα (n = 19, 50.0%), ßG‐Makassar/ßE,αα/αα (n = 4, 10.5%), ßG‐Makassar/ßNewYork,αα/αα (n = 1, 2.6%), ßG‐Makassar/ß,αα/‐α (n = 11, 28.9%), ßG‐Makassar/ß,αα/αAdanaα (n = 2, 5.3%) and ßG‐Makassar/ß,αα/–SEA (n = 1, 2.6%). The ßG‐Makassar/ß,αα/αα showed that features of thalassaemia trait with mean ± SD for Hb, MCV and MCH were 13.74 g/dL ± 2.40, 76.18 ± 6.02 fL and 25.79 ± 2.41 pg, respectively. This is the largest study reporting a significant number of Hb G‐Makassar in Malaysia. Although the mutation is similar to Hb S, the phenotype is benign.

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Section: Discussionmentioning

confidence: 99%

Clinical and haematological characteristics of 38 individuals with Hb G‐Makassar in Malaysia

Esa,

Mohamad,

Hamzah

et al. 2023

eJHaem

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“…For inherited disorders this includes early achievement of treatment end points by CTX001 treatment of β-hemoglobinopathies in clinical trials ( Frangoul et al, 2020 ) and many other preclinical and clinical evaluations ( Koniali et al, 2021 ; Cleared et al, 2022 ), and for cancer treatment this includes clinical application of autologous CAR-T cells and allogeneic CAR-NK and CAR-T cells ( Ottaviano et al, 2022 ; Valeri et al, 2022 ; Zhang et al, 2022 ). Much of this progress is based on ex vivo delivery by electroporation, which when suitably optimized achieves great efficiencies even in HSPCs as cells recalcitrant to transfection ( Hoban et al, 2015 ; Frangoul et al, 2021 ; Chu et al, 2021 ). In clinically relevant cells, DNA delivery is marked by low editing efficiencies and high toxicity, whereas protein- or RNP-based delivery allows high efficiencies but is cost- and labor-intensive.…”

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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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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“…Furthermore, an investigational new drug application was filed for BEAM-102, which was designed to change the point mutation in HBB from gTg to gCg. The result is a switch from glutamic acid to alanine in position 6, which converts HbS into a better tolerated HbG-Makassar ( 46 ).…”

Section: Genome Editing Clinical Trials For Scdmentioning

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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Conversion of HbS to Hb G-Makassar By Adenine Base Editing Is Compatible with Normal Hemoglobin Function

Cited by 15 publications

References 0 publications

Clinical and haematological characteristics of 38 individuals with Hb G‐Makassar in Malaysia

Clinical and haematological characteristics of 38 individuals with Hb G‐Makassar in Malaysia

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

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

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