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

Barbarani, Gloria; Labedz, Agata

doi:10.3389/fgeed.2020.571239

Cited by 6 publications

(3 citation statements)

References 97 publications

(119 reference statements)

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“…4 ). These data above indicate that ABE8e, in addition to Cas9 and CBE, is able to effectively disrupt the BCL11A enhancer +58 locus to induce HbF level reaching or exceeding a clinical threshold (about 30% of HbF expression 29 ) required to therapeutically ameliorate hemoglobin disorders 28 .…”

Section: Resultsmentioning

confidence: 78%

Therapeutic adenine base editing of human hematopoietic stem cells

et al. 2023

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In β-thalassemia, either γ-globin induction to form fetal hemoglobin (α2γ2) or β-globin repair to restore adult hemoglobin (α2β2) could be therapeutic. ABE8e, a recently evolved adenine base editor variant, can achieve efficient adenine conversion, yet its application in patient-derived hematopoietic stem cells needs further exploration. Here, we purified ABE8e for ribonucleoprotein electroporation of β-thalassemia patient CD34+ hematopoietic stem and progenitor cells to introduce nucleotide substitutions that upregulate γ-globin expression in the BCL11A enhancer or in the HBG promoter. We observed highly efficient on-target adenine base edits at these two regulatory regions, resulting in robust γ-globin induction. Moreover, we developed ABE8e-SpRY, a near-PAMless ABE variant, and successfully applied ABE8e-SpRY RNP to directly correct HbE and IVS II-654 mutations in patient-derived CD34+ HSPCs. Finally, durable therapeutic editing was produced in self-renewing repopulating human HSCs as assayed in primary and secondary recipients. Together, these results support the potential of ABE-mediated base editing in HSCs to treat inherited monogenic blood disorders.

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Section: Resultsmentioning

confidence: 78%

Therapeutic adenine base editing of human hematopoietic stem cells

et al. 2023

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“…Lenti-viral based approaches to complement the defective adult haemoglobin with non-sickling fetal or adult haemoglobin are currently the preferred strategies, but the long-term safety issues due to insertional mutagenesis are unknown ( Pawliuk et al, 2001 ; Ribeil et al, 2017 ; Kanter et al, 2022 ). CRISPR/Cas based approaches are now gaining recognition as ex vivo gene editing tools owing to the efficiency and versatility ( Barbarani et al, 2020 ; Prasad et al, 2021 ). Previous studies have attempted the correction of HbS mutation using Homology Directed Repair (HDR) with Cas9 but suffered from low efficiency and engraftment potential ( DeWitt et al, 2016 ).…”

Section: Introductionmentioning

confidence: 99%

Efficient and error-free correction of sickle mutation in human erythroid cells using prime editor-2

et al. 2022

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Sickle cell anaemia (SCA) is one of the common autosomal recessive monogenic disorders, caused by a transverse point mutation (GAG > GTG) at the sixth codon of the beta-globin gene, which results in haemolytic anaemia due to the fragile RBCs. Recent progress in genome editing has gained attention for the therapeutic cure for SCA. Direct correction of SCA mutation by homology-directed repair relies on a double-strand break (DSB) at the target site and carries the risk of generating beta-thalassaemic mutations if the editing is not error-free. On the other hand, base editors cannot correct the pathogenic SCA mutation resulting from A > T base transversion. Prime editor (PE), the recently described CRISPR/Cas 9 based gene editing tool that enables precise gene manipulations without DSB and unintended nucleotide changes, is a viable approach for the treatment of SCA. However, the major limitation with the use of prime editing is the lower efficiency especially in human erythroid cell lines and primary cells. To overcome these limitations, we developed a modular lenti-viral based prime editor system and demonstrated its use for the precise modelling of SCA mutation and its subsequent correction in human erythroid cell lines. We achieved highly efficient installation of SCA mutation (up to 72%) and its subsequent correction in human erythroid cells. For the first time, we demonstrated the functional restoration of adult haemoglobin without any unintended nucleotide changes or indel formations using the PE2 system. We also validated that the off-target effects mediated by the PE2 system is very minimal even with very efficient on-target conversion, making it a safe therapeutic option. Taken together, the modular lenti-viral prime editor system developed in this study not only expands the range of cell lines targetable by prime editor but also improves the efficiency considerably, enabling the use of prime editor for myriad molecular, genetic, and translational studies.

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“…Some of us started on this project during or after the first lockdown, causing further delays, which led to a multiphasic pattern of recruitment (Figure 1). During the first lockdown, we individually set up our strategies to progress our research projects at home by either reading scientific literature, writing review papers, [6][7][8][9] or planning experiments for when we would eventually return to work. By summer 2020, many of us began to slowly get back to the laboratory in shifts or for a limited time to avoid overcrowding of spaces.…”

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confidence: 99%