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β-Thalassemias are inherited anemias that are caused by the absent or insufficient production of the β chain of hemoglobin. Here we report 6-8-year follow-up of four adult patients with transfusion-dependent β-thalassemia who were infused with autologous CD34 + cells transduced with the TNS9.3.55 lentiviral globin vector after reduced-intensity conditioning (RIC) in a phase 1 clinical trial ( NCT01639690) . Patients were monitored for insertional mutagenesis and the generation of a replication-competent lentivirus (safety and tolerability of the infusion product after RIC-primary endpoint) and engraftment of genetically modified autologous CD34 + cells, expression of the transduced β-globin gene and post-transplant transfusion requirements (efficacy-secondary endpoint). No unexpected safety issues occurred during conditioning and cell product infusion. Hematopoietic gene marking was very stable but low, reducing transfusion requirements in two patients, albeit not achieving transfusion independence. Our findings suggest that non-myeloablative conditioning can achieve durable stem cell engraftment but underscore a minimum CD34 + cell transduction requirement for effective therapy. Moderate clonal expansions were associated with integrations near cancer-related genes, suggestive of non-erythroid activity of globin vectors in stem/progenitor cells. These correlative findings highlight the necessity of cautiously monitoring patients harboring globin vectors., (© 2022. The Author(s), under exclusive licence to Springer Nature America, Inc.)

The β-thalassemias are inherited blood disorders that result from insufficient production of the β-chain of hemoglobin. More than 200 different mutations have been identified. β-Thalassemia major requires life-long transfusions. The only cure for severe β-thalassemia is to provide patients with hematopoietic stem cells. Globin gene therapy promises a curative autologous stem cell transplantation without the immunologic complications of allogeneic transplantation. The future directions of gene therapy include enhancement of lentiviral vector-based approaches, fine tuning of the conditioning regimen, and the design of safer vectors. Progress in genetic engineering bodes well for finding a cure for severe globin disorders., (Copyright © 2018 Elsevier Inc. All rights reserved.)

The beta-thalassemias are inherited anemias caused by mutations that severely reduce or abolish expression of the beta-globin gene. Like sickle cell disease, a related beta-globin gene disorder, they are ideal candidates for performing a genetic correction in patient hematopoietic stem cells (HSCs). The most advanced approach utilizes complex lentiviral vectors encoding the human β-globin gene, as first reported by May et al. in 2000. Considerable progress toward the clinical implementation of this approach has been made in the past five years, based on effective CD34+ cell mobilization and improved lentiviral vector manufacturing. Four trials have been initiated in the United States and Europe. Of 16 evaluable subjects, 6 have achieved transfusion independence. One of them developed a durable clonal expansion, which regressed after several years without transformation. Although globin lentiviral vectors have so far proven to be safe, this occurrence suggests that powerful insulators with robust enhancer-blocking activity will further enhance this approach. The combined discovery of Bcl11a-mediated γ-globin gene silencing and advances in gene editing are the foundations for another gene therapy approach, which aims to reactivate fetal hemoglobin (HbF) production. Its clinical translation will hinge on the safety and efficiency of gene targeting in true HSCs and the induction of sufficient levels of HbF to achieve transfusion independence. Altogether, the progress achieved over the past 15 years bodes well for finding a genetic cure for severe globin disorders in the next decade.

Globin gene therapy requires abundant numbers of highly engraftable, autologous hematopoietic stem cells expressing curative levels of β-globin on differentiation. In this study, CD34+ cells from 31 thalassemic patients mobilized with hydroxyurea+granulocyte colony-stimulating factor (G-CSF), G-CSF, Plerixafor, or Plerixafor+G-CSF were transduced with the TNS9.3.55 β-globin lentivector and compared for transducibility and globin expression in vitro, as well as engraftment potential in a xenogeneic model after partial myeloablation. Transduction efficiency and vector copy number (VCN) averaged 48.4 ± 2.8% and 1.91 ± 0.04, respectively, whereas expression approximated the one-copy normal β-globin output. Plerixafor+G-CSF cells produced the highest β-globin expression/VCN. Long-term multilineage engraftment and persistent VCN and vector expression was encountered in all xenografted groups, with Plerixafor+G-CSF-mobilized cells achieving superior short-term engraftment rates, with similar numbers of CD34+ cells transplanted. Overall, Plerixafor+G-CSF not only allows high CD34+ cell yields but also provides increased β-globin expression/VCN and enhanced early human chimerism under nonmyeloablative conditions, thus representing an optimal graft for thalassemia gene therapy., (© 2015 by The American Society of Hematology.)

We conducted a pilot trial to investigate the safety and effectiveness of mobilizing CD34(+) hematopoietic progenitor cells (HPCs) in adults with β-thalassemia major. We further assessed whether thalassemia patient CD34(+) HPCs could be transduced with a globin lentiviral vector under clinical conditions at levels sufficient for therapeutic implementation. All patients tolerated granulocyte colony-stimulating factor well with minimal side effects. All cell collections exceeded 8 × 10(6) CD34(+) cells/kg. Using clinical grade TNS9.3.55 vector, we demonstrated globin gene transfer averaging 0.53 in 3 validation runs performed under current good manufacturing practice conditions. Normalized to vector copy, the vector-encoded β-chain was expressed at a level approximating normal hemizygous protein output. Importantly, stable vector copy number (0.2-0.6) and undiminished vector expression were obtained in NSG mice 6 months posttransplant. Thus, we validated a safe and effective procedure for β-globin gene transfer in thalassemia patient CD34(+) HPCs, which we will implement in the first US trial in patients with severe inherited globin disorders. This trial is registered at www.clinicaltrials.gov as #NCT01639690.

Realizing the therapeutic potential of human induced pluripotent stem (iPS) cells will require robust, precise and safe strategies for genetic modification, as cell therapies that rely on randomly integrated transgenes pose oncogenic risks. Here we describe a strategy to genetically modify human iPS cells at 'safe harbor' sites in the genome, which fulfill five criteria based on their position relative to contiguous coding genes, microRNAs and ultraconserved regions. We demonstrate that ∼10% of integrations of a lentivirally encoded β-globin transgene in β-thalassemia-patient iPS cell clones meet our safe harbor criteria and permit high-level β-globin expression upon erythroid differentiation without perturbation of neighboring gene expression. This approach, combining bioinformatics and functional analyses, should be broadly applicable to introducing therapeutic or suicide genes into patient-specific iPS cells for use in cell therapy.

Globin gene transfer in autologous hematopoietic stem cells offers a potentially curative treatment option for patients suffering from beta-thalassemia major who lack an HLA-matched hematopoietic stem cell donor. Based on extensive preclinical investigation, we are initiating a phase I clinical trial using G-CSF mobilized, autologous CD34(+) cells transduced with a vector similar to the original TNS9 vector. Our first mobilizations in adult beta-thalassemic subjects have been well tolerated and yielded the required CD34(+) cell dose. To minimize toxicity to enrolled subjects, and in the absence of a demonstrated requirement for myeloablative conditioning, our trial will use a reduced intensity conditioning regimen. Because low vector titers may adversely affect efficacy and safety, we have focused on vector manufacturing processes. We are now in a position to transfer our globin lentiviral vectors in a clinically relevant dosage (averaging 0.8 vector copy per cell in bulk CD34(+) cells) and to supply clinical grade vector to collaborating centers in the U.S.A. and in Europe. We anticipate that the first U.S. trial of globin gene transfer will start in 2010.

The beta-thalassemias are genetic disorders that are caused by the absent or insufficient production of the beta-chain of hemoglobin. This deficiency causes ineffective erythropoiesis and hemolytic anemia. Without treatment, the severe form of the disease is lethal within the first decade of life. The only curative therapeutic option to date is allogeneic bone marrow transplantation from a matched, related donor, which carries a low risk of morbidity and mortality. Most patients, however, lack a matched donor and are thus managed with palliative therapy, consisting of lifelong transfusion therapy combined with pharmacological chelation to curb iron accumulation. Despite a major improvement in the chelation therapy and supportive care, the major cause of death in these patients is cardiac failure due to secondary hemochromatosis. The goal of globin gene therapy is to offer a potentially curative treatment to patients lacking a matched, related donor, based on the transfer of a regulated beta-globin gene in autologous CD34+ hematopoietic cells collected following G-CSF mobilization. Our clinical trial at Memorial Sloan-Kettering Cancer Center builds on a 20-year long investigation to develop an erythroid-specific vector to regulate beta-globin transgene expression in the progeny of transduced hematopoietic stem cells. To minimize the risks to the patient, the genetically modified cells will be infused after extensive biosafety testing of the transduced cells and following the administration of a reduced intensity (non-myeloablative) conditioning regimen. The protocol will be offered to patients with transfusion-dependent ss-thalassemia who are 15 years or older and lack a matched, related donor.

beta-Thalassaemia major is a congenital anaemia for which there is presently no curative therapy other than allogeneic haematopoietic stem cell transplantation. This therapeutic option, however, is not available to most subjects for whom there is no available human leucocyte antigen-matched bone marrow donor. The transfer of a regulated globin gene in autologous haematopoietic stem cells is therefore a directly needed alternative treatment. This strategy, simple in principle, raises major challenges in terms of controlling transgene expression, which ideally should be erythroid-specific, differentiation- and stage-restricted, elevated, position independent, and sustained over time. Using lentiviral vectors, it has been demonstrated that an optimized combination of proximal and distal transcriptional control elements permits lineage-specific and elevated beta-globin expression in vivo, resulting in the correction of anaemia and secondary organ damage in beta-thalassaemic mice. Several groups have extended these findings to various models of beta-thalassaemia and sickle cell disease. Different globin vectors, however, do not express beta-globin at the same level, and accordingly require the delivery of markedly different vector copy numbers to correct anaemia. Insulators are under investigation to assess whether they might enhance globin gene expression or vector safety. Altogether, recent advances in globin vector design bode well for upcoming clinical trials to assess the therapeutic value of globin gene transfer.

Globin gene transfer in autologous hematopoietic stem cells is a promising therapeutic option for subjects with beta-thalassemia major. In this approach, high level, erythroid-specific globin transgene expression should correct ineffective erythropoiesis and hemolytic anemia following the delivery of only 1 to 2 vector copies per cell. The generation of vectors that provide high-level globin expression and require low vector copy (VC) integration is therefore essential for both safety and efficacy. We show here the major roles played by 2 lesser-known locus control region elements, termed HS1 and HS4. Partial deletions within HS4 markedly reduce in vivo globin expression requiring multiple VC per cell to correct the anemia. Most strikingly, addition of HS1 to HS2-3-4 increases globin expression by 52%, yielding 9 g Hb/VC in beta-thalassemic mice. Thus, while vectors encoding HS2-3-4 provide curative levels of hemoglobin at 1 to 2 copies per cell, adding HS1 is a promising alternative strategy if upcoming clinical trials prove higher levels of expression to be necessary.

Purpose of Review: The beta-thalassemias and sickle cell anemia are severe congenital anemias for which there is presently no curative therapy other than allogeneic hematopoietic stem cell transplantation. This therapeutic option, however, is not available to most patients due to the lack of an HLA-matched bone marrow donor. The transfer of a regulated globin gene in autologous hematopoietic stem cells is therefore a highly attractive alternative treatment. This strategy, simple in principle, raises major challenges in terms of controlling transgene expression, which ideally should be erythroid specific, differentiation and stage restricted, elevated, position independent, and sustained over time., Recent Findings: Using lentiviral vectors, May et al. demonstrated that an optimized combination of proximal and distal transcriptional control elements permits lineage-specific and elevated beta-globin expression in vivo, resulting in therapeutic hemoglobin production and correction of anemia in beta-thalassemic mice. Several groups have extended these findings to various models of beta-thalassemia and sickle cell disease. While the addition of the wild-type beta-globin gene is naturally suited for treating beta-thalassemia, several alternatives have been proposed for the treatment of sickle cell disease, using either gamma or mutant beta-globin gene addition, trans-splicing or RNA interference., Summary: These recent advances bode well for the clinical investigation of stem cell-based gene therapy in the severe hemoglobinopathies.

The beta-thalassemias are congenital anemias that are caused by mutations that reduce or abolish expression of the beta-globin gene. They can be cured by allogeneic hematopoietic stem cell (HSC) transplantation, but this therapeutic option is not available to most patients. The transfer of a regulated beta-globin gene in autologous HSCs is a highly attractive alternative treatment. This strategy, which is simple in principle, raises major challenges in terms of controlling expression of the globin transgene, which ideally should be erythroid specific, differentiation- and stage-restricted, elevated, position independent, and sustained over time. Using lentiviral vectors, May et al. demonstrated in 2000 that an optimized combination of proximal and distal transcriptional control elements permits lineage-specific and elevated beta-globin expression, resulting in therapeutic hemoglobin production and correction of anemia in beta-thalassemic mice. Several groups have by now replicated and extended these findings to various mouse models of severe hemoglobinopathies, thus fueling enthusiasm for a potential treatment of beta-thalassemia based on globin gene transfer. Current investigation focuses on safety issues and the need for improved vector production methodologies. The safe implementation of stem cell-based gene therapy requires the prevention of the formation of replication-competent viral genomes and minimization of the risk of insertional oncogenesis. Importantly, globin vectors, in which transcriptional activity is highly restricted, have a lesser risk of activating oncogenes in hematopoietic progenitors than non-tissue-specific vectors, by virtue of their late-stage erythroid specificity. As such, they provide a general paradigm for improving vector safety in stem cell-based gene therapy.

Patients affected by beta-thalassemia major require lifelong transfusions because of insufficient or absent production of the beta chain of hemoglobin (Hb). A minority of patients are cured by allogeneic bone marrow transplantation. In the most severe of the hitherto available mouse models of beta-thalassemia, a model for human beta-thalassemia intermedia, we previously demonstrated that globin gene transfer in bone marrow cells is curative, stably raising Hb levels from 8.0-8.5 to 11.0-12.0 g/dL in long-term chimeras. To fully assess the therapeutic potential of gene therapy in the context of a lethal anemia, we now have created an adult model of beta(0)-thalassemia major. In this novel model, mice engrafted with beta-globin-null (Hbb(th3/th3)) fetal liver cells succumb to ineffective erythropoiesis within 60 days. These mice rapidly develop severe anemia (2-4 g/dL), massive splenomegaly, extramedullary hematopoiesis (EMH), and hepatic iron overload. Remarkably, most mice (11 of 13) treated by lentivirus-mediated globin gene transfer were rescued. Long-term chimeras with an average 1.0-2.4 copies of the TNS9 vector in their hematopoietic and blood cells stably produced up to 12 g/dL chimeric Hb consisting of mu alpha(2):hu beta(2) tetramers. Pathologic analyses indicated that erythroid maturation was restored, while EMH and iron overload dramatically decreased. Thus, we have established an adult animal model for the most severe of the hemoglobinopathies, Cooley anemia, which should prove useful to investigate both genetic and pharmacologic treatments. Our findings demonstrate the remarkable efficacy of lentivirus-mediated globin gene transfer in treating a fulminant blood disorder and strongly support the efficacy of gene therapy in the severe hemoglobinopathies.

The beta-thalassemias are caused by more than 200 mutations that reduce or abolish beta-globin production. The severity of the resulting anemia can lead to lifelong transfusion dependency. A genetic treatment based on globin gene transfer would require that transgene expression be erythroid specific, elevated, and sustained over time. We report here that long-term synthesis of chimeric hemoglobin (mualpha(2):hubeta(A)(2)) could be achieved in mice with beta-thalassemia intermedia following engraftment with bone marrow cells transduced with a lentiviral vector encoding the human beta-globin gene. In the absence of any posttransduction selection, the treated chimeras exhibit durably increased hemoglobin levels without diminution over 40 weeks. Ineffective erythropoiesis and extramedullary hematopoiesis (EMH) regress, as reflected by normalization of spleen size, architecture, hematopoietic colony formation, and disappearance of liver EMH. These findings establish that a sustained increase of 3 to 4 g/dL hemoglobin is sufficient to correct ineffective erythropoiesis. Hepatic iron accumulation is markedly decreased in 1-year-old chimeras, indicating persistent protection from secondary organ damage. These results demonstrate for the first time that viral-mediated globin gene transfer in hematopoietic stem cells effectively treats a severe hemoglobin disorder.

The stable introduction of a functional gene into autologous stem cells is a potentially powerful approach to treat a number of inherited or acquired diseases. One challenge facing this approach is to express adequate levels of the therapeutic transgene in a regulated and sustained fashion, eventually restricting expression to a single lineage developing from the transduced stem cells. Until now, low-level expression, position effects, and transcriptional silencing have hampered the effectiveness of retroviral-mediated gene transfer. In an effort to overcome these obstacles, we have systematically investigated vectors encoding the human beta-globin gene linked to selected combinations of proximal and distal genetic regulatory elements. Our results demonstrate that with thoughtful vector design one can successfully express long-term, therapeutic levels of virally encoded human beta-globin in the erythroid progeny of hematopoietic stem cells.

The stable introduction of a functional beta-globin gene in haematopoietic stem cells could be a powerful approach to treat beta-thalassaemia and sickle-cell disease. Genetic approaches aiming to increase normal beta-globin expression in the progeny of autologous haematopoietic stem cells might circumvent the limitations and risks of allogeneic cell transplants. However, low-level expression, position effects and transcriptional silencing hampered the effectiveness of viral transduction of the human beta-globin gene when it was linked to minimal regulatory sequences. Here we show that the use of recombinant lentiviruses enables efficient transfer and faithful integration of the human beta-globin gene together with large segments of its locus control region. In long-term recipients of unselected transduced bone marrow cells, tetramers of two murine alpha-globin and two human betaA-globin molecules account for up to 13% of total haemoglobin in mature red cells of normal mice. In beta-thalassaemic heterozygous mice higher percentages are obtained (17% to 24%), which are sufficient to ameliorate anaemia and red cell morphology. Such levels should be of therapeutic benefit in patients with severe defects in haemoglobin production.