The β-hemoglobinopathies, β-thalassemia (β-thal) and sickle cell disease (SCD), are the most common monogenic diseases all over the world. They are caused by mutations in the β-globin (HBB) gene locus that result in the production of insufficient (β0, β+) or aberrant (βS) β-globin protein. In β-thal, profound anemia results from absent or insufficient hemoglobin (Hb) concentration within red blood cells (RBCs) and the toxic effects of unpaired free β-globin for RBC membranes (hemichrome precipitation) that cause ineffective erythropoiesis and chronic hemolysis. The most severe clinical form of β-thal is transfusion dependent β-thal (TDT), when patients rely on chronic RBC transfusions for the prevention of lethal complications and survival. The molecular basis for SCD is the S mutation, a single base substitution (A to T) in the sixth exon of the HBB gene that leads to Glu6Val amino-acid substitution. Sickle hemoglobin (HbS) polymerizes upon deoxygenation, reducing RBC deformability and modifying their adhesion properties, finally leading to intensely painful vaso-occlusive crises (VOC) and acute chest syndrome (ACS), irreversible organ damage, poor quality of life and reduced life expectancy. Severe SCD genotypes are represented by heterozygous βS/β0 and homozygous βS/βS.
Although both transfusion and iron chelation treatments have significantly improved over the years and, thereby improving the quality of life, they do not provide a definitive cure, since they do not address the inherent genetic cause. Thus, hematopoietic stem cell (HSC) transplantation is the only presently available feasible therapeutic approach. Nevertheless, the high success rate of allogeneic hematopoietic stem cell transplantation (HSCT), the transplantation of genetically corrected, autologous HSCs may be a curative therapeutic option for patients who lack an HLA-matched sibling donor. It avoids the need for a matched donor and thus avoids the risk of graft versus host disease and graft rejection after HSCT. Moreover, the conditioning regimen required for the engraftment of genetically modified cells, due to their autologous origin, does not include immunosuppressive drugs. The rapid worldwide application of this therapeutic approach may now be possible, because of better safety and the absence of treatment-related mortality in gene therapy trials up to now.
Figure 1. Novel Therapeutic Approaches for β-Hemoglobinopathies. (Cavazzana M, et al. 2017)
The conditions of a clinical-grade gene therapy vector can be summarized as follows: (1) controlled transgene expression: erythroid-specific, stage-restricted, position-independent, elevated, and sustained over time; (2) effective targeting of HSCs; (3) highly efficient and stable transduction; (4) absent or low genomic toxicity; and (5) correction of the phenotype in preclinical models. Research on gene therapy for hemoglobinopathies started around 30 years ago. The idea was to engineer integrating retroviruses by replacing their genes with the transgene of interest. Firstly, pioneering studies used γ-retroviral vectors (γ-RVs) derived from the Moloney leukemia virus (MLV) to successfully transfer genes into murine repopulating stem/progenitor cell; but, levels of β-globin expression were low and non-therapeutic, and variegation of gene expression was observed. The discovery of the β-globin LCR (guaranteeing high-level, erythroid-specific expression of the β-like globin genes) was fundamental in improving the design and therapeutic potential of these gene therapy vectors. However, the introduction of the LCR elements and the β-globin gene into MLV-based vectors was associated with proviral rearrangements. The introduction of HIV-derived lentiviral vectors (LVs) constituted a breakthrough, because of the LVs' ability to accommodate complex transcriptional units and efficiently transduce HSCs. Moreover, the use of self-inactivating vectors has eliminated the risk of transactivation of the adjacent cellular genes by the viral cis-regulatory elements. Most importantly, hot spots of MLV-based γ-RVs are highly enriched in proto-oncogenes, whereas LVs have a safer integration profile.
Two breakthroughs have enabled efficient HSC transduction and the therapeutic expression of β-like globin transgenes in HSC-derived RBCs: (1) the development of HIV-1-derived lentiviral vectors and (2) the discovery of the LCR HSs capable of boosting β-like globin expression. The introduction of critical LCR HS enhancers into the vector and the optimization of the vector titer resulted in proof-of-concept studies in murine models of β-thalassemia and SCD. Subsequently, multiple laboratories have shown that LVs carrying the human β-or γ-globin gene under the control of the β-globin promoter and LCR elements are able to rescue murine and human β-thalassemic and SCD phenotypes.
In the early 2000s, two groups designed the first prototype LVs: TNS9 and HPV569 both contained large fragments of the human β-globin gene and LCR HSs. These vectors can correct disease features in transgenic mouse models of β-thalassemia and SCD, respectively. The HPV569 vector was used in the first human clinical trial approved worldwide for the gene therapy of TDT and SCD. After the LG001 clinical trial, the vector was further optimized: in the BB305 transfer vector, the 5' LTR promoter was replaced by the cytomegalovirus promoter (in order to boost viral production), and the cHS4 was removed. Three Phase I/II clinical trials with the BB305 vector were approved: HGB-204 in the USA, Australia, and Thailand (for TDT), HGB-205 in France (for both TDT and SCD), and HGB-206 in the USA (for SCD).
Figure 2. Lentiviral vector-based strategies for treating β-hemoglobinopathies. (Magrin E, et al. 2019)
In the USA, the other two Phase I/II protocols for SCD have been initiated. Malik used an LV encoding a modified γ-globin transgene to treat two βS/β0 patients after reduced-intensity conditioning. The early results appear to be encouraging and the safety profile is excellent. In the second clinical trial, the vector contained a β-globin gene with three antisickling mutations. However, no results have yet been reported. Although the comparison of different gene addition clinical trials is often difficult, the data as a whole strongly show that the optimized drug manufacturing process, cell dose, patient conditioning, and HSC source represent key requirements for obtaining clinical benefit in gene therapy for β-thalassemia and SCD.
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1. Magrin E, et al. Lentiviral and genome-editing strategies for the treatment of β-hemoglobinopathies. Blood, 2019, 134(15): 1203-1213.
2. Cavazzana M, et al. Gene therapy for β-hemoglobinopathies. Molecular Therapy, 2017, 25(5): 1142-1154.
3. Dong A C, Rivella S. Gene addition strategies for β-thalassemia and sickle cell anemia. Gene and Cell Therapies for Beta-Globinopathies. Springer, New York, NY, 2017: 155-176.
4. Sii-Felice K, et al. Hemoglobin disorders: lentiviral gene therapy in the starting blocks to enter clinical practice. Experimental hematology, 2018, 64: 12-32.