Gene Therapy for β-Hemoglobinopathies
Authors: Marina Cavazzana, Chiara Antoniani, Annarita Miccio
Journal: Molecular Therapy (2017)

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. 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. Hemoglobin (Hb), the protein that carries oxygen from the lungs to the tissues, is a tetramer composed of two α-like globin chains and two β-like globin chains. In humans, β-like globin genes are sequentially expressed throughout development (Figure 1). Around the time of birth, fetal Aγ- and Gγ-globin expression is silenced, and the adult β-globin gene is predominantly expressed; this process is referred as γ-to-β Hb switching.1Sankaran V.G. Orkin S.H. The switch from fetal to adult hemoglobin.Cold Spring Harb. Perspect. Med. 2013; 3: a011643Crossref Scopus (186) Google Scholar A 16-kb-long locus control region (LCR; located ∼40–60 kb upstream of the β-globin genes) contains powerful chromatin opening and DNA enhancer elements (DNase hypersensitive sites [HSs]), which are necessary for high-level globin gene expression. The LCR is thought to regulate the expression of the β-globin-like genes by direct interaction with their promoters. The LCR loops to the β-like globin promoters in a developmentally dynamic manner and is juxtaposed to the fetal γ-globin and adult β-globin promoters in fetal and adult erythroblasts, respectively. A pentameric complex (including GATA1, TAL1, E2A, LMO2, and LDB1) is thought to mediate formation of the loop between the LCR and the globin promoters and, as a consequence, β-like globin gene expression (Figure 1). Several nuclear factors are thought to contribute to the Hb switching process, including the erythroid Kruppel-like factor (KLF1),2Borg J. Papadopoulos P. 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Wang X. Maeda M. Canver M.C. Sher F. Funnell A.P. Fisher C. Suciu M. Martyn G.E. Norton L.J. et al.Transcription factors LRF and BCL11A independently repress expression of fetal hemoglobin.Science. 2016; 351: 285-289Crossref PubMed Scopus (204) Google Scholar recently identified LRF/ZBTB7A transcription factor as a potent repressor of fetal β-like globin expression in adult human erythroid cells. LRF exerts its repressive activity through the NuRD repressor complex,13Masuda T. Wang X. Maeda M. Canver M.C. Sher F. Funnell A.P. Fisher C. Suciu M. Martyn G.E. Norton L.J. et al.Transcription factors LRF and BCL11A independently repress expression of fetal hemoglobin.Science. 2016; 351: 285-289Crossref PubMed Scopus (204) Google Scholar independently of the HbF repressor BCL11A (Figure 2). Lastly, chromatin modifiers and remodeling factors (e.g., EHMT1/2,14Renneville A. Van Galen P. Canver M.C. McConkey M. Krill-Burger J.M. Dorfman D.M. Holson E.B. Bernstein B.E. Orkin S.H. Bauer D.E. 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The region contains a 250-bp polypyrimidine-rich sequence, the deletion of which resulted in delayed human γ-to-β-globin switching in transgenic mice,19O'Neill D. Yang J. Erdjument-Bromage H. Bornschlegel K. Tempst P. Bank A. Tissue-specific and developmental stage-specific DNA binding by a mammalian SWI/SNF complex associated with human fetal-to-adult globin gene switching.Proc. Natl. Acad. Sci. USA. 1999; 96: 349-354Crossref PubMed Scopus (79) Google Scholar and SNPs associated with elevated HbF levels.20Akinsheye I. Alsultan A. Solovieff N. Ngo D. Baldwin C.T. Sebastiani P. Chui D.H. Steinberg M.H. Fetal hemoglobin in sickle cell anemia.Blood. 2011; 118: 19-27Crossref PubMed Scopus (317) Google Scholar, 21Lessard S. Beaudoin M. Benkirane K. Lettre G. Comparison of DNA methylation profiles in human fetal and adult red blood cell progenitors.Genome Med. 2015; 7: 1Crossref PubMed Scopus (35) Google Scholar Importantly, the 3.5-kb δγ-intergenic region is occupied by BCL11A in human adult primary erythroblasts, suggesting a direct role of BCL11A in γ-globin repression.9Sankaran V.G. Xu J. Byron R. Greisman H.A. Fisher C. Weatherall D.J. Sabath D.E. Groudine M. Orkin S.H. Premawardhena A. Bender M.A. A functional element necessary for fetal hemoglobin silencing.N. Engl. J. Med. 2011; 365: 807-814Crossref PubMed Scopus (133) Google Scholar This region is included in the 7.2-kb δβ-Corfu region, the deletion of which results in a high HbF blood content in thalassemic patients.22Chakalova L. Osborne C.S. Dai Y.F. Goyenechea B. Metaxotou-Mavromati A. Kattamis A. Kattamis C. Fraser P. The Corfu deltabeta thalassemia deletion disrupts gamma-globin gene silencing and reveals post-transcriptional regulation of HbF expression.Blood. 2005; 105: 2154-2160Crossref PubMed Scopus (74) Google Scholar Cross-comparisons of point mutations carried by sickle cell disease (SCD) and thalassemic patients displaying HPFH traits have revealed a common T-to-C substitution at position −175 (−175T > C) in both the Aγ- and Gγ-globin promoters responsible for pancellular expression of high levels of γ-globin.23Wienert B. Funnell A.P. Norton L.J. Pearson R.C. Wilkinson-White L.E. Lester K. Vadolas J. Porteus M.H. Matthews J.M. Quinlan K.G. Crossley M. Editing the genome to introduce a beneficial naturally occurring mutation associated with increased fetal globin.Nat. 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Nakamura Y. Hughes J.R. Hardison R.C. Blobel G.A. Li C. Weiss M.J. A genome-editing strategy to treat β-hemoglobinopathies that recapitulates a mutation associated with a benign genetic condition.Nat. Med. 2016; 22: 987-990Crossref PubMed Scopus (222) Google Scholar (M.J. Weiss and J.K. Joung, 2016, American Society of Hematology, conference) (Figure 2). These data suggest that BCL11A binding sites in the β-globin locus (e.g., in the 3.5-kb δγ-intergenic region and in the γ-globin promoter) may be responsible for HbF silencing in a redundant fashion. SCD and β-thalassemia are the most common monogenic disorders worldwide, with ∼317,000 affected neonates born each year.12Brendel C. Guda S. Renella R. Bauer D.E. Canver M.C. Kim Y.J. Heeney M.M. Klatt D. Fogel J. Milsom M.D. et al.Lineage-specific BCL11A knockdown circumvents toxicities and reverses sickle phenotype.J. Clin. Invest. 2016; 126: 3868-3878Crossref PubMed Scopus (108) Google Scholar β-Thalassemia is caused by more than 200 different β-globin gene mutations that reduce or abrogate production of the β-globin chains (the β+ and β0 genotypes, respectively). The excess of α-globin chains results in ineffective erythropoiesis, intramedullary apoptosis of erythroid precursors, and hemolytic anemia. Patients with β-thalassemia intermedia and minor can survive without regular blood transfusions. Conversely, patients affected by β-thalassemia major are transfusion dependent while they suffer from severe anemia, chronic hemolysis, iron overload, hepatosplenomegaly, and complications such as cardiopathies and endocrine disorders. The age at which these complications appear is tightly linked to the adequacy of supportive care. SCD is caused by an A-to-T point mutation in the sixth codon of the β-globin gene, which leads to an E6V substitution in the β-globin polypeptide. The resulting defective β-globin (βS) polymerizes in the deoxygenated state, and the normally flexible, doughnut-shaped red blood cells (RBCs) take on an inflexible hook/sickle shape. Sickled RBCs obstruct microvessels and have a short half-life due to repeated sickling-unsickling cycles. The pathophysiology of SCD differs from that of β-thalassemia. In fact, SCD is a chronic disease that dramatically impairs the function of many organs, independently of the transfusion and chelation regimens adopted. SCD is associated with substantial morbidity, poor quality of life, and a shortened life expectancy, even in high-income countries like France.25Ngo S. Bartolucci P. Lobo D. Mekontso-Dessap A. Gellen-Dautremer J. Noizat-Pirenne F. Audard V. Godeau B. Galacteros F. Habibi A. Causes of death in sickle cell disease adult patients: old and new trends.Blood. 2014; 124: 2715Crossref Google Scholar SCD-associated morbidity and mortality in young adults is largely due to currently unpreventable complications, such as priapism, avascular necrosis, chronic pulmonary impairment, hypertension, stroke, and recurrent veno-occlusive crises.26Madigan C. Malik P. Pathophysiology and therapy for haemoglobinopathies. Part I: sickle cell disease.Expert Rev. Mol. Med. 2006; 8: 1-23Crossref Google Scholar Survival has improved significantly over the last two decades, and 94% of children with SCD now survive until the age of 18 years thanks to better monitoring, Pneumococcus vaccination, penicillin prophylaxis, and treatment with hydroxyurea, an anti-cancer drug, which increases blood HbF levels.13Masuda T. Wang X. Maeda M. Canver M.C. Sher F. Funnell A.P. Fisher C. Suciu M. Martyn G.E. Norton L.J. et al.Transcription factors LRF and BCL11A independently repress expression of fetal hemoglobin.Science. 2016; 351: 285-289Crossref PubMed Scopus (204) Google Scholar Indeed, the clinical course of SCD is improved when fetal γ-globin genes are highly expressed,27Powars D.R. Weiss J.N. Chan L.S. Schroeder W.A. Is there a threshold level of fetal hemoglobin that ameliorates morbidity in sickle cell anemia?.Blood. 1984; 63: 921-926Crossref PubMed Google Scholar as seen in patients with HPFH traits. In SCD, γ-globin exerts a potent anti-sickling function by competing with the sickle β-globin for incorporation in the Hb tetramers and by inhibiting sickle hemoglobin (HbS) polymerization (Figure 3). However, pharmacological treatments increasing HbF levels are not equally effective in all patients, and toxicity remains a problem. In fact, mortality is still high once patients reach adulthood.14Renneville A. Van Galen P. Canver M.C. McConkey M. Krill-Burger J.M. Dorfman D.M. Holson E.B. Bernstein B.E. Orkin S.H. Bauer D.E. Ebert B.L. EHMT1 and EHMT2 inhibition induces fetal hemoglobin expression.Blood. 2015; 126: 1930-1939Crossref PubMed Scopus (72) Google Scholar The ever-increasing demand for lifelong transfusions is impossible to satisfy (for safety reasons in developing countries and for availability reasons in high-income countries). Allogenic transplantation of HSCs (HSCT) is currently the only definitive treatment for β-hemoglobinopathies. However, only a small percentage of patients can undergo this procedure, because (1) it has to be performed in specialized units and (2) the majority of the populations affected by these diseases are poorly represented in the international registry of HSC donors. Hence, in the absence of an HLA-geno-compatible sibling, the vast majority of patients lack a well-matched, unrelated donor. Gene therapy using autologous, genetically modified HSCs is an alternative to allogenic HSCT for treating β-hemoglobinopathies. It circumvents the need for a matched donor and thus avoids the risk of graft versus host disease and graft rejection after HSCT. Furthermore, the conditioning regimen required for the engraftment of genetically modified cells, because of their autologous origin, does not include immunosuppressive drugs. Hence, the infusion of the genetically modified cells and patient follow-up can potentially be performed in many pediatric and adult hematopoietic cell transplantation units, even those with limited expertise in allogenic HSCT. The rapid worldwide application of this therapeutic approach may now be possible, thanks to better safety and the absence of treatment-related mortality in gene therapy trials to date. The source of autologous CD34+ hematopoietic stem/progenitor cells (HSPCs) is critical for obtaining an adequate dose of cells retaining stem cell properties. The conventional collection of autologous bone marrow (via multiple punctures of the iliac crests under general anesthesia) is invasive and does not yield enough CD34+ HSPCs, particularly in β-thalassemic and SCD patients. In β-thalassemic bone marrow, ineffective erythropoiesis is characterized by (1) accelerated erythroid differentiation, (2) a maturation block at the polychromatophilic stage, and (3) elevated death of erythroid precursors.28Ribeil J.A. Arlet J.B. Dussiot M. Moura I.C. Courtois G. Hermine O. Ineffective erythropoiesis in β -thalassemia.Sci. World J. 2013; 2013: 394295Crossref Scopus (86) Google Scholar The consequence of these alterations is the accumulation of five to six times more erythroid precursors than in normal bone marrow; this contributed to the initial failure to harvest an adequate dose of HSPCs from β-thalassemic patient bone marrow.29Cavazzana-Calvo M. Payen E. Negre O. Wang G. Hehir K. Fusil F. Down J. Denaro M. Brady T. Westerman K. et al.Transfusion independence and HMGA2 activation after gene therapy of human β-thalassaemia.Nature. 2010; 467: 318-322Crossref PubMed Scopus (1037) Google Scholar Therefore, in these patients, mobilization of HSPCs is recommended for the gene therapy procedure.30Karponi G. Psatha N. Lederer C.W. Adair J.E. Zervou F. Zogas N. Kleanthous M. Tsatalas C. Anagnostopoulos A. Sadelain M. et al.Plerixafor+G-CSF-mobilized CD34+ cells represent an optimal graft source for thalassemia gene therapy.Blood. 2015; 126: 616-619Crossref PubMed Scopus (36) Google Scholar, 31Boulad F. Wang X. Qu J. Taylor C. Ferro L. Karponi G. Bartido S. Giardina P. Heller G. Prockop S.E. et al.Safe mobilization of CD34+ cells in adults with β-thalassemia and validation of effective globin gene transfer for clinical investigation.Blood. 2014; 123: 1483-1486Crossref PubMed Scopus (54) Google Scholar, 32Yannaki E. Karponi G. Zervou F. Constantinou V. Bouinta A. Tachynopoulou V. Kotta K. Jonlin E. 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Bellio L. et al.Plerixafor and G-CSF combination mobilizes hematopoietic stem and progenitors cells with a distinct transcriptional profile and a reduced in vivo homing capacity compared to Plerixafor alone.Haematologica. 2016; (Published online December 29, 2016)https://doi.org/10.3324/haematol.2016.154740Crossref PubMed Scopus (22) Google Scholar Similarly, two or three bone marrow harvests are required to collect an adequate dose of HSPCs from SCD patients; this is mainly because the HSC harvest is negatively influenced by (1) the inflammatory SCD bone marrow microenvironment34Zhang D. Xu C. Manwani D. Frenette P.S. Neutrophils, platelets, and inflammatory pathways at the nexus of sickle cell disease pathophysiology.Blood. 2016; 127: 801-809Crossref PubMed Scopus (245) Google Scholar and (2) the formation of cell aggregates during the isolation of bone marrow mononuclear cells (M.C., unpublished data). In individuals with SCD, the administration of G-CSF led to severe adverse events35Fitzhugh C.D. Hsieh M.M. Bolan C.D. Saenz C. Tisdale J.F. Granulocyte colony-stimulating factor (G-CSF) administration in individuals with sickle cell disease: time for a moratorium?.Cytotherapy. 2009; 11: 464-471Abstract Full Text Full Text PDF PubMed Scopus (87) Google Scholar and is therefore contraindicated. We recently suggested that plerixafor is potentially a safer mobilizing agent for SCD patients (http://www.clinicaltrials.gov identifier NCT02212535). Lastly, the choice of the optimal conditioning regimen is particularly important, since the intake of a high proportion of transduced HSCs is required to ameliorate the clinical phenotype of β-thalassemia and SCD; the selective advantage is at best limited to the preferential survival of corrected erythroblasts. Several approaches can be used to genetically manipulate HSCs and correct the genetic defect underlying β-hemoglobinopathies. A number of ongoing clinical trials (based on the transplantation of autologous β-thalassemic and SCD HSCs) are investigating the use of lentiviral vectors that express β-like globin transgenes (G. Ferrari, 2016, Mol Ther., conference).29Cavazzana-Calvo M. Payen E. Negre O. Wang G. Hehir K. Fusil F. Down J. Denaro M. Brady T. Westerman K. et al.Transfusion independence and HMGA2 activation after gene therapy of human β-thalassaemia.Nature. 2010; 467: 318-322Crossref PubMed Scopus (1037) Google Scholar, 36Cavazzana M. Gene therapy studies in hemoglobinopathies: successes and challenges.Blood. 2016; 128 (SCI–50)PubMed Google Scholar, 37Kanter J. Walters M.C. Hsieh M.M. Krishnamurti L. Kwiatkowski J. Kamble R.T. von Kalle C. Kuypers F.A. Cavazzana M. Leboulch P. et al.Interim results from a phase 1/2 clinical study of lentiglobin gene therapy for severe sickle cell disease.Blood. 2016; 128: 1176Crossref Google Scholar, 38Thompson A.A. Kwiatkowski J.L. Rasko J. Hongeng S. Schiller G.J. 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