Search bioRxiv⌕ Search

Biology subjects

Chalumeau, A.

Publications and source records attributed to Chalumeau, A..

5 recordsLinked to original sources
bioRxiv2026

Enhanced γ-globin reactivation and sickle cell correction through a repressor-to- activator motif switch in the HBG1/2 promoters

Sickle cell disease (SCD) is caused by the production of an abnormal adult hemoglobin that generates sickle-shaped red blood cells (RBCs). Transplantation of autologous genetically corrected hematopoietic stem/progenitor cells (HSPCs) represents a promising therapy. Persistent fetal hemoglobin expression improves SCD. Here, we engineered the fetal HBG1/2 promoters by replacing the BCL11A repressor binding site (BS) with a TAL1:GATA1 motif recognized by transcriptional activators. We exploited the prime editing nuclease (PEn) that efficiently installed the TAL1:GATA1 motif in K562 cells, outperforming the original PE. Non-homologous end joining (NHEJ) and/or alternative-end joining (alt-EJ) pathway inhibition enhanced precise editing. However, this strategy was poorly efficient in patients HSPCs. Alternatively, we used CRISPR/Cas9 nuclease to either disrupt the BCL11A BS via NHEJ and/or alt-EJ or to replace it with the TAL1:GATA1 motif via homology-directed repair (HDR) using a donor ssODN template. NHEJ and alt-EJ inhibition improved product purity, reducing InDels and achieving superior precise editing efficiency compared to PEn in K562 and HSPCs. HDR-edited HSPCs preserved clonogenic capacity and differentiated into RBCs showing elevated HBG expression and correction of the sickling phenotype. These results demonstrate that replacing the BCL11A BS with a TAL1:GATA1 motif is a potent strategy for reactivating HBG1/2 to treat SCD.

bioengineering↗
bioRxiv2024

Multiplex base editing of BCL11A regulatory elements to treat sickle cell disease

Sickle cell disease (SCD) is a genetic anemia caused by the production of an abnormal adult hemoglobin. The clinical severity is lessened by elevated fetal hemoglobin (HbF) production in adulthood. A promising therapy is the transplantation of autologous, hematopoietic stem/progenitor cells (HSPCs) treated with CRISPR/Cas9 to downregulate the HbF repressor BCL11A via generation of double strand breaks (DSBs) in the +58-kb erythroid-specific enhancer. Here, to further enhance HbF production without increasing the mutagenic load, we targeted both +58-kb and +55-kb BCL11A erythroid-specific enhancers using base editors. We systematically dissected DNA motifs recognized by the key transcriptional activators within these regions and identified the critical nucleotides required for activator binding. Multiplex base editing of these residues was efficient and safe and generated no or little DSBs and genomic rearrangements. We observed substantial HbF reactivation, exceeding the levels achieved using the CRISPR/Cas9 nuclease-based strategy, thus efficiently rescuing the sickling phenotype. Multiplex base editing was efficient in long-term repopulating HSPCs and resulted in potent HbF reactivation in vivo. In summary, these results show that multiplex base editing of BCL11A erythroid-specific enhancers is a safe and potent strategy for treating sickle cell disease.

molecular biology↗
bioRxiv2024

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

Reactivation of fetal hemoglobin (HbF) expression through clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9-mediated disruption of regulatory elements involved in {gamma}-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 {gamma}-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 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 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.

bioengineering↗
bioRxiv2022

Novel lentiviral vectors for gene therapy of sickle cell disease combining gene addition and gene silencing strategies

Sickle cell disease (SCD) is due to a mutation in the {beta}-globin (HBB) gene causing the production of the toxic sickle hemoglobin (HbS, a2{beta}S2). Transplantation of autologous hematopoietic stem/progenitor cells (HSPCs) transduced with lentiviral vectors (LVs) expressing an anti-sickling {beta}-globin ({beta}AS) is a promising treatment; however, it is only partially effective and patients still present elevated HbS levels. Here, we developed a bifunctional LV expressing {beta}AS3-globin and an artificial microRNA (amiR) specifically downregulating {beta}S-globin expression with the aim of reducing HbS levels and favoring {beta}AS3 incorporation into Hb tetramers. Efficient transduction of SCD HSPC by the bifunctional LV led to a substantial decrease of {beta}S-globin transcripts in HSPC-derived erythroid cells, a significant reduction of HbS+ red cells and effective correction of the sickling phenotype, outperforming {beta}AS gene addition and BCL11A gene silencing strategies. The bifunctional LV showed a standard integration profile and neither the HSPC viability, engraftment and multi-lineage differentiation nor the erythroid transcriptome and miRNAome were affected by the treatment, confirming the safety of this therapeutic strategy. In conclusion, the combination of gene addition and gene silencing strategies can improve the efficacy of current LV-based therapeutic approaches without increasing the mutagenic vector load, thus representing a novel treatment for SCD.

genetics↗