Gene Therapy for β-Hemoglobinopathies

Abstract

β-Thalassemia and sickle cell disease (SCD) are the world's two most widely disseminated hereditary hemoglobinopathies. β-Thalassemia originated in the Mediterranean, Middle Eastern, and Asian regions, and SCD originated in central Africa. However, subsequent population migration means that these two diseases are now global and thus constitute a growing health problem in many countries. Despite remarkable improvements in medical care for patients with β-hemoglobinopathies, there is still only one definitive treatment option: allogeneic hematopoietic stem cell (HSC) transplantation. The development of gene therapy for β-hemoglobinopathies has been justified by (1) the limited availability of human leukocyte antigen (HLA)-identical donors, (2) the narrow window of application of HSC transplantation to the youngest patients, and (3) recent advances in HSC-based gene therapy. The huge ongoing efforts in translational medicine and the high number of related publications show that gene therapy has the potential to become the treatment of choice for patients who lack either an HLA genoidentical sibling or an alternative, medically acceptable donor. In this dynamic scientific context, we first summarize the main steps toward clinical translation of this therapeutic approach and then discuss novel lentiviral- and genome editing-based treatment strategies for β-hemoglobinopathies.

Keywords: gene therapy; hematopoietic stem cell; hemoglobinopathies; sickle cell disease; thalassemias.

Figure 1

Developmental Regulation of the β-Globin-like Genes The human β-like globin gene locus contains five β-like globin genes. The ε-globin is expressed during the embryonic stage and replaced by γ-globin during fetal life. Around time of birth, a γ-to-β-globin switch occurs and the β-globin is predominantly expressed in the adult life. The adult δ-globin gene is poorly expressed. A pentameric complex mediates long-range interactions between the LCR and γ- and β-globin promoters in fetal and adult erythroblasts, respectively.

Figure 2

Major Players in Fetal-to-Adult Hb Switching BCL11A interacts with SOX6, GATA1, FOG1, and the NuRD repressor complex to repress the expression of γ-globin genes in adult erythroblasts. BCL11A binding sites (indicated with light blue boxes) were mapped in the γ-globin promoters and in a putative 3.5-kb HbF silencer (depicted as a black box). The expression of BCL11A is positively regulated by KLF1, which in turn is upregulated by MYB. Additionally, KLF1 favors fetal-to-adult Hb switching by directly activating β-globin gene expression. The transcription factor LRF silences γ-globin expression through the NuRD complex.

Figure 3

Inhibition of HbS Polymerization by Anti-sickling β-like Globins HbS tetramers polymerize upon de-oxygenation. In HbS polymers, the valine at position 6 (V6) of the β^S-chain forms a lateral contact with the phenylalanine and leucine residues at positions 85 and 88 (F85 and L88) of the β^S-chain of the adjacent tetramer. Additionally, a glutamic acid (at position 22) of the β-chain interacts with a histidine residue (at position 20) of the α-globin of a neighboring tetramer (axial contact). The incorporation of γ- or β^T87Q-globin into the Hb prevents HbS polymerization. β^T87Q-Globin harbors an amino acid substitution at position 87 (threonine [T87] to glutamine [Q87]). The glutamine residue derived from the γ-globin chain inhibits the formation of lateral contacts between the Hb tetramers. In addition to the T87Q amino acid substitution, in AS3 β-globin, the glutamic acid at position 22 is replaced by an alanine (A22), which disrupts the axial contacts between the Hb tetramers, and the glycine at position 16 is replaced by an aspartic acid (D16), thus increasing the affinity of the AS3 β-chain to the α-globin polypeptide.

Figure 4

Novel Therapeutic Approaches for β-Hemoglobinopathies Overview of lentiviral (1a–1c) and genome editing (2a and 2b) approaches for correcting HSCs from SCD and β-thalassemic patients. (1a) Gene addition: therapeutic, erythroid-specific expression of β-like globin transgenes using lentiviral vectors. (1b) shRNA for BCL11A knockdown (KD): HbF reactivation by lentiviral-mediated erythroid-specific expression of an shRNA against BCL11A. (1c) Forced LCR-γ-globin looping: HbF induction by lentiviral expression of a ZF-LDB1 fusion protein forcing the juxtaposition of the LCR to the γ-globin genes. (2a) Gene correction: correction of SCD and β-thalassemic mutations via nuclease-induced HDR. (2b) BCL11A enhancer disruption: HbF reactivation induced by nuclease-mediated targeting of the BCL11A erythroid-specific enhancer, located at +58 kb from BCL11A transcription start site. (2c) Generation of HPFH mutations: HbF induction by reproducing a 13-bp HPFH deletion in the γ-globin promoters via MMEJ or deletional HPFH mutations (encompassing the β- and δ-globin genes) by NHEJ.

Figure 5

β-Globin-like Expressing Lentiviral Vectors Used in Clinical Trials Schematic representation of β-like globin expressing lentiviral vectors in their proviral forms. The clinical trial number is indicated in brackets. LTRs deleted of 400 bp in the HIV U3 region (ΔLTR), rev-responsive element (RRE), splicing donor (SD), and splicing acceptor (SA) sites, human β-like globin genes, β-globin promoter (bp), the 3′ β-globin enhancer (E), and Dnase-I hypersensitive sites HS2, HS3, and HS4 from β-globin LCR are shown. Two copies of the cHS4 core and the FB insulator were inserted into the LTRs of HPV569 and Lenti-βAS3-FB lentiviral vectors, respectively.