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Gene Therapy Through Autologous Transplantation of Gene-Modified Hematopoietic Stem Cells

2012; Elsevier BV; Volume: 19; Issue: 1 Linguagem: Inglês

10.1016/j.bbmt.2012.09.021

1523-6536

Donald B. Kohn, Sung‐Yun Pai, Michel Sadelain,

Neurogenetic and Muscular Disorders Research

Various genetic diseases of blood cells have been successfully treated with allogeneic hematopoietic stem cell (HSC) transplantation (HSCT) (Table 1). Given that all mature blood cells and their progenitors are derived from HSCs, replacement of genetically defective HSCs with normal donor HSCs replenishes the hematopoietic and lymphoid systems with genetically normal blood cells.Table 1Genetic Diseases of Blood Cells Treated with Allogeneic HSCTCell TypeDiseasesRBCsβ-thalassemia, SCDWBCsLymphocyte defects: SCID, Wiskott-Aldrich syndrome, leukocyte adhesion deficiency, X-HIM, HLH, IPEXNeutrophil defects: CGD, Chediak-Higashi syndrome, Shwachman-Diamond syndromeStorage/metabolic diseases: Gaucher disease, ALD, MLD, MPS, osteopetrosisPlateletsAmegakaryocytosis, Glanzmann thrombastheniaStem/progenitor cellsFanconi anemia, Diamond-Blackfan anemia, cyclic neutropenia, Kostmann sundromeDiseases in italics have been examined in clinical gene therapy trials.X-HIM indicates X-linked immunodeficiency with hyper-IgM; HLH, Hemophagocytic Lymphohistiocytosis; IPEX, immune dysregulation, polyendocrinopathy, enteropathy, X-linked; ALD, adrenoleukodystrophy; MLD, metachromatic leukodystrophy; MPS, mucopolysaccharidosis. Open table in a new tab Diseases in italics have been examined in clinical gene therapy trials. X-HIM indicates X-linked immunodeficiency with hyper-IgM; HLH, Hemophagocytic Lymphohistiocytosis; IPEX, immune dysregulation, polyendocrinopathy, enteropathy, X-linked; ALD, adrenoleukodystrophy; MLD, metachromatic leukodystrophy; MPS, mucopolysaccharidosis. The biology of HSCs makes them amenable to transplantation. They can be isolated ex vivo from a donor by marrow harvest, peripheral blood stem cell (PBSC) mobilization, or umbilical cord blood collection; processed to enrich them from the far more abundant mature cells; manipulated; and transplanted by i.v. infusion. After transplantation, HSCs can produce new blood cells of all lineages for the life of the recipient, producing an enduring effect. HSCs are highly generative, with each stem cell producing, through the proliferation of a series of successively more lineage-restricted progenitor cells, literally millions of mature progeny cells. However, HSCs are rare in their native settings, fragile with ex vivo manipulation, and mostly quiescent, which may protect them from accumulating DNA damage but makes their genetic manipulation more difficult. Currently, it is not possible to expand the numbers of pluripotent HSCs in culture to any useful extent, although efforts aimed at doing so are ongoing. From more than 40 years of allogeneic HSCT practice, we have learned several key concepts. Transplantation outcomes vary by HSC source and disease-specific comorbidities. Results are generally better with matched sibling donors than with all other allogeneic HSCT donor options (ie, unrelated adult or cord blood donors, haploidentical family donors), owing to immunologic complications of graft-versus-host disease (GVHD) or graft rejection, which may occur more frequently, with increasing disparity for HLAs and minor antigens. Marrow and immune ablation are usually provided with high-dose combination chemotherapy regimens (eg, busulfan/cyclophosphamide or busulfan/fludarabine, with or without serotherapy, such as antithymocyte globulin). Although fully ablative conditioning generally leads to the highest levels of donor engraftment, conditioning-associated toxicities are a major cause of morbidity and mortality. Efforts to reduce the intensity of conditioning have led to lower frequencies of toxicity, but may be limited by the concurrent higher rates of late graft failure and GVHD. The success of reduced-intensity regimens for nonmalignant hematologic disease depends in part on the minimum level of lineage-specific donor cells required for correction of clinical symptoms, which varies for different diseases. HSCT is the standard of care for life-threatening primary immune deficiencies, such as severe combined immune deficiency (SCID), which is uniformly fatal without transplantation. Since the late 1960s, HSCT has been performed in patients with SCID using matched sibling donors without previous conditioning with >90% survival at most centers [1Buckley R.H. Transplantation of hematopoietic stem cells in human severe combined immunodeficiency: long-term outcomes.Immunol Res. 2011; 49: 25-43Crossref PubMed Scopus (148) Google Scholar, 2Hassan A. Booth C. Brightwell A. et al.Outcome of hematopoietic stem cell transplantation for adenosine deaminase-deficient severe combined immunodeficiency.Blood. 2012; Jul 12; 120: 3615-3624Crossref PubMed Scopus (136) Google Scholar, 3Fernandes J.F. Rocha V. Labopin M. et al.Eurocord and Inborn Errors Working Party of European Group for Blood and Marrow Transplantation. Transplantation in patients with SCID: mismatched related stem cells or unrelated cord blood?.Blood. 2012; 119: 2949-2955Crossref PubMed Scopus (90) Google Scholar]. SCID is the first disease for which matched unrelated donor (MUD) bone marrow (1974) and haploidentical (parental) bone marrow (1976) were used in HSCT. Outcomes of HSCT using donors other than matched sibling donors have continued to improve over time but remain worse than outcomes with matched sibling donors, owing to immunologic reactions, delayed immune reconstitution, and in many cases, the use of conditioning therapy before transplantation. Typical disease-free survival rates after allogeneic HSCT range from 65% to 75%, although there is a great deal of variability based on approach and center experience. Given the limitations of allogeneic HSCT for patients with SCID lacking a matched sibling donor, gene therapy was first investigated for SCID, and SCID is the first disorder for which gene therapy was shown to be clinically efficacious. Sickle cell disease (SCD) has also been treated with allogeneic HSCT, although at a much lower rate than for SCID. Patients with SCD present several additional challenges to successful allogeneic HSCT. They are often alloimmunized as a result of frequent transfusions, which may further increase the risk of rejection over that in patients with an otherwise normal immune system. They may also have SCD-related organ impairments, which increase risks associated with complications of conditioning and GVHD. In previously reported series, matched sibling donor HSCT for SCD was successful in ∼90% of cases, with a 5%-10% rate of nonengraftment and SCD recurrence and a 5%-10% rate of transplantation-related mortality [4Walters M.C. Sullivan K.M. Stem-cell transplantation for sickle cell disease.N Engl J Med. 2010; 362: 955-956Crossref PubMed Scopus (10) Google Scholar, 5Hsieh M.M. Kang E.M. Fitzhugh C.D. et al.Allogeneic hematopoietic stem-cell transplantation for sickle cell disease.N Engl J Med. 2009; 361: 2309-2317Crossref PubMed Scopus (318) Google Scholar]. HSCT is performed less often in patients with SCD lacking a matched sibling donor, because of the increased risks of immunologic rejection and GVHD. Outcomes are continually improving for all types of allogeneic HSCT in response to better donor matching, conditioning regimens, immune manipulations, and clinical management strategies. Nevertheless, there remains significant room for improvement in the safety and efficacy of HSCT, to provide the benefits of this therapy to the majority of people affected by genetic blood cell diseases. The central hypothesis of gene therapy for genetic blood cell diseases is that HSCT using gene-modified autologous HSCs has similar benefits as allogeneic HSCT, with fewer immunologic complications. Effective gene modification of HSCs must be permanent to allow the introduced correction to persist as the HSCs proliferate and produce their abundant progeny. Efforts to date have focused on stable addition of a replacement gene (cDNA, β-globin mini-locus, or genomic segment) or in situ modification of the endogenous gene. The primary approach has been the integration of viral vectors of the Retroviridae family that covalently insert their passenger gene into the chromosome of the target HSCs. Transfection or electroporation of plasmids or use of adenoviral or AAV vectors for HSC modification has yielded low efficiency and mostly transient gene presence, and in some cases possibly prohibitive cytotoxicity. In theory, an autonomous gene cassette that is able to self-replicate fast enough to be maintained in the cytoplasm of cells as they proliferate (eg, an episomal plasmid or artificial chromosome) may be used for HSC gene therapy, but none of suitable activity has yet been produced. Retroviridae that have been used for stable gene transfer to HSCs include the γ-retroviral vectors (typically derived from the murine leukemia virus family), lentiviral vectors (typically derived from HIV-1), and spumaviruses (typically the human foamy virus). All of these viruses can be turned into gene delivery vectors by removing the viral genes encoding their structural and enzymatic proteins and replacing them with the human transgene of interest. The viral proteins needed for virion production can be provided in trans during a packaging step, resulting in vectors that can enter target cells once to integrate their genome payload, but are incapable of producing new viruses in the target cell, absolutely preventing viral replication and ongoing infection. The past 2-3 decades have brought incremental improvements in gene delivery technology, with vectors made at higher titers and clinical-scale production methods established, in parallel with the identification of optimal cytokines and culture methods for the process of HSC transduction with vectors. These vectors can in many cases lead to gene transfer to the majority of human HSCs being treated. Ideally, every HSC would be modified with a single copy of the corrective gene; however, Poisson distribution dictates that efforts to increase the percentage of HSCs that are transduced lead to an increased number of vector copies per transduced cell, which may add a risk of genotoxicity. Thus, efficient and controlled gene modification of HSCs remains one of the central technical challenges to advancing effective gene therapy for many conditions in which high percentages of gene-corrected HSCs are required for clinical benefit. Gene modification of a patient's HSCs entails relatively complex, patient-specific cell processing after HSC procurement (through bone marrow harvest, PBSC mobilization with leukapheresis, or cord blood collection). Performed under current Good Manufacturing Practice conditions, the HSCs are usually enriched by CD34 immunoaffinity selection, then cultured for several days in a mixture of stimulating cytokines (eg, ckit ligand, flt-3 ligand, thrombopoietin) on an extracellular matrix protein (eg, recombinant fibronectin) layer, with one or more additions of the gene delivery vector to the cells after a prestimulation phase. The cells are then formulated for i.v. infusion by multiple cell washes and suspension in a medium such as plasmalyte. Before being readministered, the cell product must meet predefined release criteria for viability and absence of microbiological contamination. Further characterization (eg, colony formation, vector copy number by quantitative PCR, transgene product expression) is done postinfusion on reserved aliquots of the final cell product. These procedures have been established at multiple academic sites and are well within the capabilities of clinical stem cell processing laboratories that have current Good Manufacturing Practice procedures and environments in place. The initial attempts at autologous HSCT using gene therapy (performed between approximately 1992 and 2006) were directed against primary immune deficiencies, including adenosine deaminase (ADA)-deficient SCID, X-linked SCID (XSCID), chronic granulomatous disease (CGD), and leukocyte adhesion deficiency. These trials used γ-retroviral vectors to deliver the appropriate cDNA to HSCs with the retroviral long terminal repeats (LTRs) containing strong enhancers and promoters to drive transgene expression. ADA deficiency was identified as a genetic cause of some cases of human SCID (∼15%) in the early 1970s, and normal human ADA cDNA was cloned in the 1980s as retroviral vector technology was developing, making this a logical first candidate disease for gene therapy. Initial trials of gene therapy for ADA SCID using HSCs were performed in Italy, the Netherlands, and the United States in the early 1990s, targeting CD34+ cells from bone marrow and umbilical cord blood ∗These studies followed the initial clinical trial of gene therapy for ADA-deficient SCID performed at the National Institutes of Health in 1990 in which peripheral blood T cells were transduced. [6Bordignon C. Notarangelo L.D. Nobili N. et al.Gene therapy in peripheral blood lymphocytes and bone marrow for ADA-immunodeficient patients.Science. 1995; 270: 470-475Crossref PubMed Scopus (644) Google Scholar, 7Hoogerbrugge P.M. van Beusechem V.W. Fischer A. et al.Bone marrow gene transfer in three patients with adenosine deaminase deficiency.Gene Ther. 1996; 3: 179-183PubMed Google Scholar, 8Kohn D.B. Weinberg K.I. Nolta J.A. et al.Engraftment of gene-modified umbilical cord blood cells in neonates with adenosine deaminase deficiency.Nat Med. 1995; 1: 1017-1023Crossref PubMed Scopus (505) Google Scholar]. No cytoreductive conditioning was applied in these first studies, possibly because the selective survival and amplification advantage of ADA-replete lymphocytes might have been sufficient to allow clinically beneficial immune reconstitution with only low-level engraftment of gene-corrected HSCs. In retrospect, relatively poor gene transfer to HSCs was obtained with the culture conditions used at the time, and gene-corrected blood and marrow cells were detected in subjects at only very low levels for short periods, with no clinical effects realized. Besides the incremental improvements in vector production and CD34+ cell transduction conditions that were developed subsequently, the major advance in gene therapy for ADA-deficient SCID was made by a group in Milan that administered a nonmyeloablative dose of busulfan (4 mg/kg) to subjects before reinfusion of HSCs corrected by an ADA gene retroviral vector [9Aiuti A. Slavin S. Aker M. et al.Correction of ADA-SCID by stem cell gene therapy combined with nonmyeloablative conditioning.Science. 2002; 296: 2410-2413Crossref PubMed Scopus (981) Google Scholar]. This relatively low dose of busulfan, which was well tolerated clinically, increased engraftment of gene-corrected HSCs to allow reconstitution of T and B cell numbers and function and conferred protective immunity, which has persisted for more than a decade to date [10Aiuti A. Cattaneo F. Galimberti S. et al.Gene therapy for immunodeficiency due to adenosine deaminase deficiency.N Engl J Med. 2009; 360: 447-458Crossref PubMed Scopus (816) Google Scholar]. Subsequent trials using the approach of nonmyeloablative conditioning before reinfusion of retroviral vector-transduced bone marrow CD34+ cells performed in the United Kingdom and the United States reported similar clinical outcomes, with best effects seen in patients with ADA-SCID treated in infancy [11Gaspar H.B. Cooray S. Gilmour K.C. et al.Hematopoietic stem cell gene therapy for adenosine deaminase-deficient severe combined immunodeficiency leads to long-term immunological recovery and metabolic correction.Sci Transl Med. 2011; 3: 97ra80Crossref PubMed Google Scholar, 12Candotti F. Shaw K.L. Muul L. et al.Gene therapy for adenosine deaminase-deficient severe combined immune deficiency: clinical comparison of retroviral vectors and treatment plans.Blood. 2012; 120: 3635-3646Crossref PubMed Scopus (194) Google Scholar]. To date, a total of 40 subjects have been treated in the 3 trials, all of whom survived and most of whom attained protective immune function as a result of the gene therapy. There have been no instances of vector-related complications in any of these patients, in sharp contrast to the results seen in patients with other primary immune deficiency diseases, for reasons not well understood. It can be stated that this approach may be considered as a standard of care for ADA-deficient patients lacking a matched sibling donor, although the total numbers of treated subjects and duration of follow-up remain relatively low. The most common genetic form of human SCID is X-linked (XSCID), caused by functional absence of the common cytokine receptor gamma chain (γc), which was cloned in the mid-1990s. French investigators constructed retroviral vectors carrying a normal human γc cDNA and initiated a clinical trial targeting bone marrow CD34+ cells, with HSCT performed without cytoreductive conditioning. As they first reported in 2000, vigorous immune reconstitution was seen in almost all subjects treated, with rapid increases in T lymphocyte numbers and restoration of protective immunity at a pace at least matching that seen with allogeneic HSCT from matched sibling donors [13Cavazzana-Calvo M. Hacein-Bey S. de Saint Basile G. et al.Gene therapy of human severe combined immunodeficiency (SCID)-X1 disease.Science. 2000; 288: 669-672Crossref PubMed Scopus (2232) Google Scholar, 14Hacein-Bey-Abina S. Hauer J. Lim A. et al.Efficacy of gene therapy for X-linked severe combined immunodeficiency.N Engl J Med. 2010; 363: 355-364Crossref PubMed Scopus (499) Google Scholar]. A similar trial conducted in the United Kingdom also found prompt T cell immune recovery, which persists to the present day [15Gaspar H.B. Cooray S. Gilmour K.C. et al.Long-term persistence of a polyclonal T cell repertoire after gene therapy for X-linked severe combined immunodeficiency.Sci Transl Med. 2011; 3: 97ra79Crossref PubMed Scopus (242) Google Scholar]. A significant complication developed in some of the subjects treated in these 2 trials, however, with 5 of 20 developing a T lymphoproliferative disorder approximately 3-5 years after gene therapy [16Hacein-Bey-Abina S. Garrigue A. Wang G.P. et al.Insertional oncogenesis in 4 patients after retrovirus-mediated gene therapy of SCID-X1.J Clin Invest. 2008; 118: 3132-3142Crossref PubMed Scopus (1395) Google Scholar]. This leukemia-like complication responded to chemotherapy in 4 of the subjects, who recovered with continued immune function from the initial gene therapy, but 1 child died as a result of the disease. Although this complication is indeed tragic, a clear-cut benefit was seen in many of the treated subjects, and disease-free survival was ∼85%, at least equivalent to that from the allogeneic transplant options available for patients lacking a matched sibling donor. Other clinical trials performed using γ-retroviral vector-mediated gene transfer to HSCs (granulocyte colony-stimulating factor–mobilized PBSCs) for the primary immune deficiencies CGD and Wiskott-Aldrich syndrome had similar mixed results, with clear-cut immune reconstitution in most subjects but leukoproliferative complications occurring in several treated subjects a few years later [17Stein S. Ott M.G. Schultze-Strasser S. et al.Genomic instability and myelodysplasia with monosomy 7 consequent to EVI1 activation after gene therapy for chronic granulomatous disease.Nat Med. 2010; 16: 198-204Crossref PubMed Scopus (631) Google Scholar, 18Boztug K. Schmidt M. Schwarzer A. et al.Stem-cell gene therapy for the Wiskott-Aldrich syndrome.N Engl J Med. 2010; 363: 1918-1927Crossref PubMed Scopus (435) Google Scholar]. The wild-type, replication-competent γ-retroviruses from which the vectors were derived are known to cause hematologic malignancies when they infect and proliferate to high levels in neonatal mice, leading to multiple rounds of integration into target cell genomes. This occurs through a process of insertional oncogenesis (IO), in which the potent transcriptional enhancer elements contained within the LTRs of the retroviruses deregulate expression of cellular genes that happen to be near their semirandom integration sites in the genome. It was postulated that this event is very rare and would not likely occur in the clinical setting, where replication-incompetent vectors would only be able to enter the genome once. Nevertheless, it is now recognized that IO may occur with the numbers of integrants achieved in the clinical setting. The cases of leukoproliferative disease occurred just as analytic methods were being developed that allowed their detailed study. The development of efficient PCR-based methods to isolate the DNA sequences at the junction between the vector provirus and the flanking cellular chromosome (eg, linear amplification-mediated (LAM)-PCR), as well as methods for high-throughput sequencing of these junctional fragments, and the decoding of the human genome sequence allowed the analysis of vector integration sites and their assignment to specific chromosomal locations even in complex mixtures of cells with thousands of different integrants [19Paruzynski A. Arens A. Gabriel R. et al.Genome-wide high-throughput integrome analyses by nrLAM-PCR and next-generation sequencing.Nat Protoc. 2010; 5: 1379-1395Crossref PubMed Scopus (136) Google Scholar, 20Brady T. Roth S.L. Malani N. et al.A method to sequence and quantify DNA integration for monitoring outcome in gene therapy.Nucleic Acids Res. 2011; 39: e72Crossref PubMed Scopus (63) Google Scholar]. Different types of retroviral vectors are now known to have characteristic integration preferences in the genome, affected by chromatin structure and cellular factors that play roles in guiding integrating vector genomes. The γ-retroviral vectors tend to integrate near the 5′ ends of genes being actively expressed; close proximity to the transcriptional control elements of the genes allows their deregulation [21Wu X. Li Y. Crise B. et al.Transcription start regions in the human genome are favored targets for MLV integration.Science. 2003; 300: 1749-1751Crossref PubMed Scopus (1140) Google Scholar]. HIV-1–based lentiviral vectors have an even higher predilection to integrate into genes being actively transcribed, although without the preference for the 5′ ends of the genes. Following these initial innate integration patterns, HSC clones with vectors integrated near to cellular genes controlling growth or survival may have these genes trans-activated by the vector and experience increased growth and begin a progression to flank transformation. Assays have been developed to quantify the potential of vectors to induce IO, mainly using murine HSCs in clonal cell culture or in bone marrow transplantation models [22Modlich U. Bohne J. Schmidt M. et al.Cell-culture assays reveal the importance of retroviral vector design for insertional genotoxicity.Blood. 2006; 108: 2545-2553Crossref PubMed Scopus (276) Google Scholar, 23Montini E. Cesana D. Schmidt M. et al.The genotoxic potential of retroviral vectors is strongly modulated by vector design and integration site selection in a mouse model of HSC gene therapy.J Clin Invest. 2009; 119: 964-975Crossref PubMed Scopus (462) Google Scholar]. From these studies, it is clear that the major factor determining the risk of a vector causing IO is the strength of the enhancer elements that it contains, because these elements may affect adjacent cellular transcription by acting on the cell gene promoter. Other, less common mechanisms of IO have been identified, including disruption of tumor-suppressor genes by insertion directly into them or truncation of cellular gene transcripts by providing ectopic splicing signals or polyadenylation signals, eliminating downstream regulatory regions, such as microRNA-binding sites [24Moiani A. Paleari Y. Sartori D. et al.Lentiviral vector integration in the human genome induces alternative splicing and generates aberrant transcripts.J Clin Invest. 2012; 122: 1653-1666Crossref PubMed Scopus (114) Google Scholar]. Stemming from this new understanding, a new generation of gene delivery vectors has been produced that do not contain the potent LTR enhancers, but rather use cellular gene promoters lacking strong enhancer activity (eg, PGK, elongation factor α, WASP). In the experimental models of IO, these new vectors exhibit significantly lower, often undetectable, ability to cause IO [25Baum C. Modlich U. Göhring G. et al.Concise review: managing genotoxicity in the therapeutic modification of stem cells.Stem Cells. 2011; 29: 1479-1484Crossref PubMed Scopus (39) Google Scholar]. New trials are currently under way or in development using these vectors to determine whether the clear-cut efficacy that had been achieved in earlier trials for primary immune deficiencies can be maintained with decreased risks for the complications of IO in XSCID, Wiskott-Aldrich syndrome, CGD, and chronic granulomatous disease. In addition, successful trials have been performed or are under way using lentiviral vectors for several lysosomal storage or metabolic disorders amenable to allogeneic HSCT, including X-adrenoleukodystrophy, metachromatic leukodystrophy, globoid leukodystrophy, and Hurler syndrome. The major hemoglobinopathies β-thalassemia and SCD have long been studied as candidates for treatment by gene therapy. These disorders pose several unique challenges. Transfer of a simple β-globin cDNA does not lead to the sufficient high-level expression needed to balance the production of α-globin to ameliorate β-thalassemia or reduce the percentage of βS in SCD. Expression of the β-globin gene has been studied extensively in transgenic mice, and studies have determined that a complex mini-gene is needed for high-level erythroid-specific expression, containing the exons and introns of the β-globin gene, 5′ and 3′ flanking regions with the transcriptional regulatory elements, and portions of a series of DNA segments lying upstream from the entire β-globin gene complex that play a role in opening the regional chromatin to allow transcription in erythroid cells (the locus-control region). When these β-globin mini-genes are inserted into the murine germline, the resulting transgenic mice express the β-globin gene at levels sufficient to correct thalassemia. Attempts by multiple investigators to carry these mini-genes in the γ-retroviral vector were met with failure, because the complex cassettes contained cryptic splice sites and transcription termination signals and the vectors underwent severe rearrangements, so that there was very little delivery of intact expression units [26Novak U. Harris E.A. Forrester W. et al.High-level β-globin expression after retroviral transfer of locus activation region-containing human β-globin gene derivatives into murine erythroleukemia cells.Proc Natl Acad Sci USA. 1990; 87: 3386-3390Crossref PubMed Scopus (105) Google Scholar]. Sadelain et al. [27May C. Rivella S. Callegari J. et al.Therapeutic haemoglobin synthesis in β-thalassaemic mice expressing lentivirus-encoded human β-globin.Nature. 2000; 406: 82-86Crossref PubMed Scopus (490) Google Scholar] first showed that HIV-1–based lentiviral vectors are capable of carrying these β-globin mini-genes intact, owing in part to the presence of the HIV-1 rev-responsive element in the vectors, which allows nuclear-to-cytoplasmic export of intact full-length vector genomic transcripts during packaging in the presence of the REV protein. Using β-globin lentiviral vectors, these authors demonstrated correction in murine β-thalassemic models [27May C. Rivella S. Callegari J. et al.Therapeutic haemoglobin synthesis in β-thalassaemic mice expressing lentivirus-encoded human β-globin.Nature. 2000; 406: 82-86Crossref PubMed Scopus (490) Google Scholar]. Based on this work and similar studies by other investigators, a clinical trial for β-thalassemia was initiated in France using a lentiviral vector. One subject treated demonstrated a progressive rise in hematocrit in the year after treatment, but the mechanism behind this increase turned out to be complex [28Cavazzana-Calvo M. Payen E. Negre O. et al.Transfusion independence and HMGA2 activation after gene therapy of human β-thalassaemia.Nature. 2010; 467: 318-322Crossref PubMed Scopus (1035) Google Scholar]. There was expansion of a single vector-containing hematopoietic stem or progenitor cell clone that contributed approximately one-third of the hemoglobin produced. In this clone, the vector was integrated into an intron of the HMGA2 gene and acted as a splice site and transcriptional termination sequence, truncating the HMGA2 transcript, eliminating the 3′ end of the message that conferred suppression by a microRNA. As a result of this, the HMGA2 gene was overexpressed, which may underlie the clonal expansion. This expansion has been stable over several years to the present time and did result in a therapeutic effect, but is another example of unexpected genotoxicity from inserting vectors. Several strategies have been developed for the treatment of SCD with gene therapy. Expression of normal β-globin may be beneficial in SCD by counteracting the prosickling activity of HbS, but very high expression levels are required to produce a state similar to sickle cell trait. Expression of fetal (γ-) globin can prevent sickling at much lower molar concentrations than β-globin, as seen in hereditary persistence of fetal hemoglobin. Vectors have been created in which the γ-globin coding sequences have been embedded in a β-globin expression cassette to achieve expression in adult erythroid cells [29Wilber A. Hargrove P.W. Kim Y.S. et al.Therapeutic levels of fetal hemoglobin in erythroid progeny of β-thalassemic CD34+ cells after lentiviral vector-mediated gene transfer.Blood. 2011; 117: 2817-2826Crossref PubMed Scopus (88) Google Scholar]. An alternative approach in which the key amino acids of γ-