The Beta-Globin Locus and the Hemoglobin Switching: Comparison
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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. 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 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).  

  • genome editing
  • hemoglobinopathies
  • sickle cell disease

1. Introduction

Beta-hemoglobinopathies are a group of inherited recessive disorders, caused by a wide range of mutations within the β-globin locus. It is estimated that approximately 400,000 affected births occur annually [1]. Until now, more than 300 mutations affecting the β-globin expression have been described, leading to decreased or absent β-globin production (β+ or β0 thalassemia, respectively) or the expression of a mutant variant of the β-globin (sickle cell disease).
In β-thalassemia, reduced production of β-globin chains results in an excess of misfolding alpha-globin chains (a matter of quantity) consequently causing erythrocytes’ membrane damage, early apoptosis, and ineffective erythropoiesis. The secondary consequences of these primary defects are anemia, extramedullary hematopoiesis, splenomegaly, and iron accumulation [2]. The conventional therapy of β-thalassemia includes life-long blood transfusions and iron chelation in order to prevent long-term multi-organ damages caused by iron deposition. Sickle cell disease (SCD) is caused by a point mutation in the sixth codon of the β-globin gene, causing a substitution of glutamic acid for valine and subsequent expression of an abnormal form of hemoglobin (a matter of quality), termed HbS. Under hypoxia, HbS is polymerized, generating deformed, rigid, sickle-shaped red cells (sickle cells) entrapped in microvessels. Erythrocyte, and subsequently leukocyte, entrapment in the microcirculation causes vascular obstruction and tissue ischaemia, thus resulting in painful and life-threatening acute vaso-occlusive crises (VOC) [3]. The severity of the disease depends on the co-inheritance of SCD, either with beta-thalassemia mutations that reduce total HbS production, or with the hereditary persistence of fetal hemoglobin (HPFH) that results in high HbF levels with anti-sickling properties in adult life [4]. In the same context, Hydroxyurea (hydroxycarbamide), the only FDA-approved drug for SCD treatment, prevents VOC via HbF induction [5][6].
The only established curative option for both β -thalassemia and SCD is the allogenic transplantation of hematopoietic stem cells (allo-HSCT). The major drawbacks of allo-HSCT are the requirement of an HLA-matched donor, age restrictions for a successful therapeutic outcome (≤14 years), and the necessity for long-term immunosuppression in order to prevent or treat the immunological complications associated with the procedure [7].
In the last 30 years, the development of a wide range of gene therapy approaches for β-hemoglobinopathies offers not only an alternative but also an equally effective, curative option for patients without an available HLA-identical donor [8]. Gene therapy for β-hemoglobinopathies conventionally relies on the autologous transplantation of genetically modified hematopoietic stem and progenitor cells (HSPCs). The procedure includes mobilization of CD34+ HSPCs from the bone marrow to circulation after treatment with pharmaceutical reagents such as the granulocyte-colony stimulating factor G-CSF in combination with Plerixafor (AMD3100; Mozobil™) in thalassemia patients [9][10][11], or Plerixafor alone in SCD patients [12]. Following leukapheresis, the mobilized HSPCs are genetically corrected ex vivo and re-administered to the preconditioned patient [13].
Presently, there are two leading gene therapy approaches. The first and the most well-studied approach is the addition of the therapeutic gene, which could be the β- or γ-globin gene, or a modified β-globin gene (βT87Q, βAS3) with anti-sickling properties [14][15][16][17][18]. Gene addition is a “one-size-fits-all” approach; irrespective of the underlying mutation, the normal globin gene that is added into the hematopoietic stem cells (HSCs) expresses the normal protein, and the thalassemic or sickle cell phenotype can be corrected. The insertion of the therapeutic cassette in patients’ genomes to ensure life-long gene expression usually employs an integrating lentiviral (LV) vector. To date, gene addition approaches for thalassemia and SCD have been extensively applied both in preclinical models [17][19][20] and in clinical trials with therapeutic outcomes [21][22][23]. Despite the success, there are still some limitations: (i) the need for high vector copy numbers (VCNs) in order to achieve sustained and increased transgene expression, (ii) the need for optimization of the gene transfer protocols for improved transduction efficiency, (iii) the low engraftment rate of genetically modified cells that is correlated with suboptimal therapeutic outcomes, and (iv) the potential development of insertional mutagenesis phenomena due to the semirandom integration pattern of LV vectors [24].
In contrast to the “one-size-fits-all” gene addition approach, gene editing is a mutation-specific approach. The tremendous development of genome editing technology (e.g., ZFN, TALENs, CRISPR-Cas9) allowed for the targeted introduction of mutations in the genome in order to correct point mutations. Single-mutation diseases such as SCD, represent ideal targets for gene editing. Moreover, the ability of gene editing to introduce targeted mutations that may cause a disease-modulatory effect made it possible to use a universal approach to target, in addition to SCD, the many different thalassemia mutations through the reactivation of the endogenous γ-globin gene [25][26][27][28][29][30][31][32]. Gene editing utilizes for the targeted introduction of point mutations or small deletions and insertions of the cells’ endogenous DNA damage response mechanism. Generally, two main pathways are involved: non-homologous end joining (NHEJ) and homologous derived recombination (HDR) [33][34]. The variety of genome editing tools generates multiple therapeutic avenues, overcoming some concerns as regards gene addition with integrating viral vectors, including insertional genotoxicity and recombination events during viral production [35][36].

2. The Beta-Globin Locus and the Hemoglobin Switching

Hemoglobin, the tetrameric protein responsible for oxygen transportation from the lungs to all tissues, is formed by a symmetric pairing of a globin chain dimer consisting of two alpha-like and two beta-like globin chains. The alpha-globin locus including the α- and ζ-globin genes is located on chromosome 16, while the beta-globin locus is encoded by a gene cluster of ε-, γ-, δ-, and β-globins on chromosome 11. These globin genes are developmentally regulated leading to several forms of hemoglobin during different developmental stages. Specifically, the ζ2ε2, α2ε2, and ζ2γ2 tetramers are present during embryonic development while the α2γ2, also known as fetal hemoglobin or HbF, predominates during fetal life. The switch from fetal to adult hemoglobin, mostly α2β2 (HbA), and in lower frequency α2δ2 (HbA2), occurs shortly after birth during the neonatal period [2][37][38][39]. These developmental switches in the β-globin locus, from embryonic to fetal and fetal to adult hemoglobin, are tightly regulated by the interaction of the beta-globin locus control region (LCR) with the promoters of the respective genes (Figure 1). The beta-globin LCR consists of five DNase I hypersensitive sites (DHSs), of which HS2-4 express a cell type-specific enhancer activity, increasing the transcription levels of each of the β-globin-like genes in a developmental manner [37].
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Figure 1. Hereditary persistence of fetal hemoglobin (HPFH). (A) The beta-globin locus is located at p15.4 of chromosome 11 and consists of (B) the β- locus control region (LCR), hypersensitive sites (HS1-HS5), and beta-like genes family, including HBE1, HBG1, HBG2, BGLT3, HBBP1, HBD, and HBB. The most common HPFH-related deletional mutations caused by deletions of large genomic sequence fragments are depicted. (C) Non-deletional HPFH mutations (nucleotide substitutions or small deletion) are located within the promoters of HBG1 and/or HBG2. Highlighted are the sequences recognized and bound by the transcriptional HbF regulators; LRF (red), and BCL11A (blue).
Normally, HbF expression in adult life is reduced to 1%, however, in some cases, fetal hemoglobin expression persists at higher levels. This benign condition is usually caused by mutations in the β-gene cluster or the γ-promoter gene region and is known as the hereditary persistence of fetal hemoglobin (HPFH). HPFH is generally classified into two categories: deletional and non-deletional. Deletional HPFH is characterized by large deletions (~13–85 kb) in the regions between the β- and γ-globin genes. The majority of these large deletions lead to the loss of δ-globin, effectively altering the dynamics of LCR interaction with γ- and β- genes, and/or the ψβ pseudogene which seems to include a hemoglobin switching-related regulatory element [40][41][42]. Non-deletional HPFH is caused by point mutations or small deletions within both γ-globin HBG1 (Aγ) and HBG2 (Gγ) promoters. Some of these SNPs either disrupt transcription suppressors’ binding sites or create de novo binding sites for HG1/HBG2 transcription activators. For instance, the single nucleotide substitution such as −114 C > T, −117 G > A, 13 bp deletion [Δ13 bp] and −195 C > G, −196 C > T, −197 C > T, −201 C > T, −202 C > T disrupt binding sites of two major HbF silencers, BCL11A and ZBTB7A, respectively [41]. In contrast, specific point substitutions such as −113 A > G, −175 T > C, and −198 T > C create de novo binding sites for the erythroid activators GATA1, TAL1, and KLF1, respectively, that are involved in globin regulation [43][44][45][46] (Figure 1). Co-inheritance of HPFH with β-thalassemia or sickle cell disease ameliorates the severity of the disease and reduces or abrogates blood transfusions or SCD-associated complications [47][48]. The development of genome editing tools has granted the possibility to either attempt correction of the disease mutations or to mimic naturally occurring HPFH or HPFH-like mutations in order to reactivate the silenced γ-globin.

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