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Sickle cell disease and β-thalassemia affect the production of the adult β-hemoglobin chain. The clinical severity is lessened by mutations that cause fetal γ-globin expression in adult life (i.e., the hereditary persistence of fetal hemoglobin). Mutations clustering ~200 nucleotides upstream of the HBG transcriptional start sites either reduce binding of the LRF repressor or recruit the KLF1 activator. Here, we use base editing to generate a variety of mutations in the −200 region of the HBG promoters, including potent combinations of four to eight γ-globin-inducing mutations. Editing of patient hematopoietic stem/progenitor cells is safe, leads to fetal hemoglobin reactivation and rescues the pathological phenotype. Creation of a KLF1 activator binding site is the most potent strategy – even in long-term repopulating hematopoietic stem/progenitor cells. Compared with a Cas9-nuclease approach, base editing avoids the generation of insertions, deletions and large genomic rearrangements and results in higher γ-globin levels. Our results demonstrate that base editing of HBG promoters is a safe, universal strategy for treating β-hemoglobinopathies. Antoniou and colleagues used base editing to generate a variety of mutations inducing γ-globin and rescue the β-hemoglobinopathy phenotype. This strategy was safe and effective in long-term repopulating hematopoietic stem/progenitor cells.

In sickle cell disease (SCD), the β6 Glu→Val substitution in the β-globin leads to red blood cell sickling. The transplantation of autologous, genetically modified hematopoietic stem and progenitor cells (HSPCs) is a promising treatment option for patients with SCD. We completed a Phase I/II open-label clinical trial (NCT03964792) for patients with SCD using a lentiviral vector (DREPAGLOBE) expressing a potent anti-sickling β-globin. The primary endpoint was to evaluate the short-term safety and secondary endpoints included the efficacy and the long-term safety. We report on the results after 18 to 36 months of follow-up. No drug-related adverse events or signs of clonal hematopoiesis were observed. Despite similar vector copy numbers in the drug product, gene-marking in peripheral blood mononuclear cells and correction of the clinical phenotype varied from one patient to another. Single-cell transcriptome analyses show that in the patients with poor engraftment, the most immature HSCs display an exacerbated inflammatory signature (via IL-1 or TNF-α and interferon signaling pathways). This signature is accompanied by a lineage bias in the HSCs. Our clinical data indicates that the DREPAGLOBE-based gene therapy (GT) is safe. However, its efficacy is variable and probably depends on the number of infused HSCs and intrinsic, engraftment-impairing inflammatory alterations in HSCs. Trial: NCT03964792 The DREPAGLOBE lentiviral-based gene therapy for sickle cell disease is safe. However, its efficacy depends on the number of infused hematopoietic stem cells (HSCs) and intrinsic, engraftment impairing inflammatory alterations in HSCs.

Abstract The Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/Cas9 nuclease system has allowed the generation of disease models and the development of therapeutic approaches for many genetic and non-genetic disorders. However, the generation of large genomic rearrangements has raised safety concerns for the clinical application of CRISPR/Cas9 nuclease approaches. Among these events, the formation of micronuclei and chromosome bridges due to chromosomal truncations can lead to massive genomic rearrangements localized to one or few chromosomes. This phenomenon, known as chromothripsis, was originally described in cancer cells, where it is believed to be caused by defective chromosome segregation during mitosis or DNA double-strand breaks. Here, we will discuss the factors influencing CRISPR/Cas9-induced chromothripsis, hereafter termed CRISPRthripsis, and its outcomes, the tools to characterize these events and strategies to minimize them. Graphical Abstract A DNA double-strand break can lead to the formation of acentric chromosome fragments and micronuclei. After DNA condensation, the chromosome fragment is shattered generating multiple DNA fragments, which are reincorporated in the nuclear genome forming a chromotriptic chromosome. If not reincorporated in the genome, these fragments can be lost (deleted fragments) or form double-minute chromosomes.

Nuclease-based genome editing strategies hold great promise for the treatment of blood disorders. However, a major drawback of these approaches is the generation of potentially harmful double strand breaks (DSBs). Base editing is a CRISPR-Cas9-based genome editing technology that allows the introduction of point mutations in the DNA without generating DSBs. Two major classes of base editors have been developed: cytidine base editors or CBEs allowing C>T conversions and adenine base editors or ABEs allowing A>G conversions. The scope of base editing tools has been extensively broadened, allowing higher efficiency, specificity, accessibility to previously inaccessible genetic loci and multiplexing, while maintaining a low rate of Insertions and Deletions (InDels). Base editing is a promising therapeutic strategy for genetic diseases caused by point mutations, such as many blood disorders and might be more effective than approaches based on homology-directed repair, which is moderately efficient in hematopoietic stem cells, the target cell population of many gene therapy approaches. In this review, we describe the development and evolution of the base editing system and its potential to correct blood disorders. We also discuss challenges of base editing approaches–including the delivery of base editors and the off-target events–and the advantages and disadvantages of base editing compared to classical genome editing strategies. Finally, we summarize the recent technologies that have further expanded the potential to correct genetic mutations, such as the novel base editing system allowing base transversions and the more versatile prime editing strategy.

Myeloproliferative neoplasms (MPNs) are a group of disorders characterized by clonal expansion of abnormal hematopoietic stem cells leading to hyperproliferation of one or more myeloid lineages. The main complications in MPNs are high risk of thrombosis and progression to myelofibrosis and leukemia. MPN patients with high risk scores are treated by hydroxyurea (HU), interferon-α, or ruxolitinib, a tyrosine kinase inhibitor. Polycythemia vera (PV) is an MPN characterized by overproduction of red blood cells (RBCs). ABCG2 is a member of the ATP-binding cassette superfamily transporters known to play a crucial role in multidrug resistance development. Proteome analysis showed higher ABCG2 levels in PV RBCs compared to RBCs from healthy controls and an additional increase of these levels in PV patients treated with HU, suggesting that ABCG2 might play a role in multidrug resistance in MPNs. In this work, we explored the role of ABCG2 in the transport of ruxolitinib and HU using human cell lines, RBCs, and in vitro differentiated erythroid progenitors. Using stopped-flow analysis, we showed that HU is not a substrate for ABCG2. Using transfected K562 cells expressing three different levels of recombinant ABCG2, MPN RBCs, and cultured erythroblasts, we showed that ABCG2 potentiates ruxolitinib-induced cytotoxicity that was blocked by the ABCG2-specific inhibitor KO143 suggesting ruxolitinib intracellular import by ABCG2. In silico modeling analysis identified possible ruxolitinib-binding site locations within the cavities of ABCG2. Our study opens new perspectives in ruxolitinib efficacy research targeting cell types depending on ABCG2 expression and polymorphisms among patients.

Fetal hemoglobin (HbF) reactivation is a promising therapy for beta-hemoglobinopathies. We developed a prime editing strategy that introduces multiple mutations in the fetal gamma-globin promoters of patients' hematopoietic stem/progenitor cells (HSPCs), boosting HbF expression and offering alternative therapeutic perspectives.Competing Interest StatementThe authors have declared no competing interest.

The structure and function of the mitochondrial network are finely regulated. Among the proteins involved in these regulations, mitochondrial dynamics actors have been reported to regulate the apoptotic process. We show here in the Drosophila model that the mitophagy inducers, PINK1 (PTEN-induced putative kinase 1) and BNIP3 (Bcl-2 Interacting Protein 3), modulate mitochondrial apoptosis differently. If close links between the fission-inducing protein DRP1 and Bcl-2 family proteins, regulators of apoptosis, are demonstrated, the connection between mitophagy and apoptosis is still poorly understood. In Drosophila, we have shown that Rbf1, a homolog of the oncosuppressive protein pRb, induces cell death in proliferating larval tissues through a mechanism involving the interaction of Drp1 with Debcl, a pro-apoptotic protein of the Bcl-2 family. This interaction is necessary to induce mitochondrial fission, ROS production, and apoptosis. To better understand the interactions between the proteins involved in mitochondrial homeostasis and the apoptotic process, we focused on the role of two known players in mitophagy, the proteins PINK1 and BNIP3, during mitochondrial apoptosis induced by Rbf1 and Debcl in a proliferating Drosophila larval tissue. We show that Rbf1- or Debcl-induced apoptosis is accompanied by mitophagy. Interestingly, PINK1 and BNIP3 have distinct effects in regulating cell death. PINK1 promotes rbf1- or debcl-induced apoptosis, whereas BNIP3 protects against Rbf1-induced apoptosis but reduces Debcl-induced tissue loss without inhibiting Debcl-induced cell death. Furthermore, our results indicate that BNIP3 is required to induce basal mitophagy while PINK1 is responsible for mitophagy induced by rbf1 overexpression. These results highlight the critical role of mitophagy regulators in controlling homeostasis and cell fate.Competing Interest StatementThe authors have declared no competing interest.Footnotes* Supplementary Figure 4 replaced (did not correspond to the experiment described in the legend); Legend to Figure 5B corrected; A few points clarified; Some typographical errors corrected.

Sickle cell disease (SCD) is due to a mutation in the β-globin (HBB) gene causing the production of the toxic sickle hemoglobin (HbS, α2βS2). Transplantation of autologous hematopoietic stem/progenitor cells (HSPCs) transduced with lentiviral vectors (LVs) expressing an anti-sickling β-globin (βAS) is a promising treatment; however, it is only partially effective and patients still present elevated HbS levels. Here, we developed a bifunctional LV expressing βAS3-globin and an artificial microRNA (amiR) specifically downregulating βS-globin expression with the aim of reducing HbS levels and favoring βAS3 incorporation into Hb tetramers. Efficient transduction of SCD HSPC by the bifunctional LV led to a substantial decrease of βS-globin transcripts in HSPC-derived erythroid cells, a significant reduction of HbS+ red cells and effective correction of the sickling phenotype, outperforming βAS 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.Competing Interest StatementThe authors have declared no competing interest.

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 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.

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.Competing Interest StatementAM is named as inventor on a patent describing genome-editing approaches for hemoglobinopathies (WO/2020/053224/PCT/EP2019/074131: Methods for increasing fetal hemoglobin content in eukaryotic cells and uses thereof for the treatment of hemoglobinopathies). All other authors declare no competing interests.