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Beta-hemoglobinopathies are the most common genetic disorders worldwide, caused by a wide spectrum of mutations in the β-globin locus, and associated with morbidity and early mortality in case of patient non-adherence to supportive treatment. Allogeneic transplantation of hematopoietic stem cells (allo-HSCT) used to be the only curative option, although the indispensable need for an HLA-matched donor markedly restricted its universal application. The evolution of gene therapy approaches made possible the ex vivo delivery of a therapeutic β- or γ- globin gene into patient-derived hematopoietic stem cells followed by the transplantation of corrected cells into myeloablated patients, having led to high rates of transfusion independence (thalassemia) or complete resolution of painful crises (sickle cell disease-SCD). Hereditary persistence of fetal hemoglobin (HPFH), a syndrome characterized by increased γ-globin levels, when co-inherited with β-thalassemia or SCD, converts hemoglobinopathies to a benign condition with mild clinical phenotype. The rapid development of precise genome editing tools (ZFN, TALENs, CRISPR/Cas9) over the last decade has allowed the targeted introduction of mutations, resulting in disease-modifying outcomes. In this context, genome editing tools have successfully been used for the introduction of HPFH-like mutations both in HBG1/HBG2 promoters or/and in the erythroid enhancer of BCL11A to increase HbF expression as an alternative curative approach for β-hemoglobinopathies. The current investigation of new HbF modulators, such as ZBTB7A, KLF-1, SOX6, and ZNF410, further expands the range of possible genome editing targets. Importantly, genome editing approaches have recently reached clinical translation in trials investigating HbF reactivation in both SCD and thalassemic patients. Showing promising outcomes, these approaches are yet to be confirmed in long-term follow-up studies. Full article

We have previously demonstrated that both the original γ-globin lentiviral vector (LV) GGHI and the optimized GGHI-mB-3D LV, carrying the novel regulatory elements of the 3D HPFH-1 enhancer and the 3’ β-globin UTR, can significantly increase HbF production in thalassemic CD34⁺ cells and ameliorate the disease phenotype in vitro. In the present study, we investigated whether the GGHI-mB-3D vector can also exhibit an equally therapeutic effect, following the transduction of sickle cell disease (SCD) CD34⁺ cells at MOI 100, leading to HbF increase coupled with HbS decrease, and thus, to phenotype improvement in vitro. We show that GGHI-mB-3D LV can lead to high and potentially therapeutic HbF levels, reaching a mean 2-fold increase to a mean value of VCN/cell of 1.0 and a mean transduction efficiency of 55%. Furthermore, this increase was accompanied by a significant 1.6-fold HbS decrease, a beneficial therapeutic feature for SCD. In summary, our data demonstrate the efficacy of the optimized γ-globin lentiviral vector to improve the SCD phenotype in vitro, and highlights its potential use in future clinical SCD trials. Full article

Considerable attention has been recently devoted to mechanisms involved in the perinatal hemoglobin switch, as it was long ago established that the survival of fetal hemoglobin (HbF) production in significant amount can reduce the severity of the clinical course in severe disorders like β-thalassemia and sickle cell disease (SCD). For instance, when β-thalassemia is associated with hereditary persistence of fetal hemoglobin (HPFH) the disease takes a mild course, labeled as thalassemia intermedia. The same clinical amelioration occurs for the association between HPFH and SCD. As for the mechanism of this effect, some information has been obtained from the study of natural mutations at the human β-globin locus in patients with increased HbF, like the Corfu thalassemia mutations. Important evidence came from the discovery that drugs capable of improving the clinical picture of SCD, like decitabine ad hydroxycarbamide, are acting through the reactivation, to some extent, of HbF synthesis. The study of the mechanism of action of these compounds was followed by the identification of some genetic determinants, which promote this event. In particular, among a few genetic factors involved in this process, the most relevant appears the BCL11A gene, which is now credited to be able to silence γ-globin genes in the perinatal period by interaction with several erythroid-specific transcription factors and is actually considered as a barrier to HbF reactivation by known HbF inducing agents. Epigenetics is also a player in the process, mainly through DNA demethylation. This is certified by the recent demonstration that hypomethylating agents such as 5-azacytidine and decitabine, the first compounds used for HbF induction by pharmacology, act as irreversible inhibitors of demethyltransferase enzymes. Great interest has also been raised by the finding that several micro-RNAs, which act as negative regulators of gene expression, have been implicated in the progression of globin gene expression and, particularly, in the reactivation of γ-globin gene expression associated with increased HbF synthesis. Probably, this reactivation is achieved by post-transcriptional inhibition of BCL11A expression. Finally, attention is presently focused on a recently discovered BCL11A enhancer, essential for erythroid expression of BCL11A, which might become a therapeutic target for genome engineering in the β-hemoglobinopathies as its disruption affects only the erythropoietic lineage, without hurting other cell or tissue compartments. Full article