Abstract
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.
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Data availability
The data that support the findings of this study are available from the corresponding author upon reasonable request. All requests for raw and analyzed data and materials are promptly reviewed by the corresponding author to verify if the request is subject to any intellectual property or confidentiality obligations. Patient-related data not included in this paper were generated as part of clinical trials and might be subject to patient confidentiality. Any data and materials that can be shared will be released via a material transfer agreement.
The following databases were used for oncogene definitions: the ‘Bushman lab oncogenes database’ (http://www.bushmanlab.org/links/genelists, version 5, June 2021) and four levels of The Cancer Genome Atlas version of the OncoVar database (https://oncovar.org, version 1.2, August 2020).
Sequence data were deposited in the National Center of Biotechnology Information’s Sequence Read Archive (SRA BioProject PRJNA705203). For integration site analysis relative to Gene Ontology, the ‘GO.db’ Bioconductor annotation data package, version 3.13.0, was used.
Code availability
Source code for manuscript analysis has been deposited in an archived format in the Zenodo code base (https://doi.org/10.5281/zenodo.4569099).
References
Taher, A. T., Weatherall, D. J. & Cappellini, M. D. Thalassaemia. Lancet 391, 155–167 (2018).
Weatherall, D. J. & Clegg, J. B. The Thalassemia Syndrome (Blackwell Scientific, 1981).
Kountouris, P. et al. IthaGenes: an interactive database for haemoglobin variations and epidemiology. PLoS ONE 9, e103020 (2014).
Orkin, S. & Nathan, D. G. Hematology of Infancy and Childhood (W. B. Saunders, 1998).
Stamatoyannopoulos, G. The Molecular Basis of Blood Diseases (W.B. Saunders, 2001).
Borgna-Pignatti, C. et al. Survival and complications in patients with thalassemia major treated with transfusion and deferoxamine. Haematologica 89, 1187–1193 (2004).
Ladis, V. et al. Survival in a large cohort of Greek patients with transfusion-dependent β thalassaemia and mortality ratios compared to the general population. Eur. J. Haematol. 86, 332–338 (2011).
Mancuso, A., Sciarrino, E., Renda, M. C. & Maggio, A. A prospective study of hepatocellular carcinoma incidence in thalassemia. Hemoglobin 30, 119–124 (2006).
Sadelain, M. et al. Therapeutic options for patients with severe β-thalassemia: the need for globin gene therapy. Hum. Gene Ther. 18, 1–9 (2007).
Lucarelli, G., Isgro, A., Sodani, P. & Gaziev, J. Hematopoietic stem cell transplantation in thalassemia and sickle cell anemia. Cold Spring Harb. Perspect. Med. 2, a011825 (2012).
Baronciani, D. et al. Hematopoietic cell transplantation in thalassemia and sickle cell disease: report from the European Society for Blood and Bone Marrow Transplantation Hemoglobinopathy Registry: 2000–2017. Blood 132, 168 (2018).
Fitzhugh, C. D., Abraham, A. & Hsieh, M. M. Alternative donor/unrelated donor transplants for the β-thalassemia and sickle cell disease. Adv. Exp. Med. Biol. 1013, 123–153 (2017).
Weatherall, D. J. The challenge of haemoglobinopathies in resource-poor countries. Br. J. Haematol. 154, 736–744 (2011).
Mansilla-Soto, J., Riviere, I., Boulad, F. & Sadelain, M. Cell and gene therapy for the β-thalassemias: advances and prospects. Hum. Gene Ther. 27, 295–304 (2016).
Ferrari, G., Thrasher, A. J. & Aiuti, A. Gene therapy using haematopoietic stem and progenitor cells. Nat. Rev. Genet. 22, 216–234 (2020).
Magrin, E., Miccio, A. & Cavazzana, M. Lentiviral and genome-editing strategies for the treatment of β-hemoglobinopathies. Blood 134, 1203–1213 (2019).
Drysdale, C. M. et al. Hematopoietic-stem-cell-targeted gene-addition and gene-editing strategies for β-hemoglobinopathies. Cell Stem Cell 28, 191–208 (2021).
Persons, D. A. & Tisdale, J. F. Gene therapy for the hemoglobin disorders. Semin. Hematol. 41, 279–286 (2004).
Sadelain, M. Recent advances in globin gene transfer for the treatment of β-thalassemia and sickle cell anemia. Curr. Opin. Hematol. 13, 142–148 (2006).
May, C. et al. Therapeutic haemoglobin synthesis in β-thalassaemic mice expressing lentivirus-encoded human β-globin. Nature 406, 82–86 (2000).
May, C., Rivella, S., Chadburn, A. & Sadelain, M. Successful treatment of murine β-thalassemia intermedia by transfer of the human β-globin gene. Blood 99, 1902–1908 (2002).
Rivella, S., May, C., Chadburn, A., Riviere, I. & Sadelain, M. A novel murine model of Cooley anemia and its rescue by lentiviral-mediated human β-globin gene transfer. Blood 101, 2932–2939 (2003).
Lisowski, L. & Sadelain, M. Locus control region elements HS1 and HS4 enhance the therapeutic efficacy of globin gene transfer in β-thalassemic mice. Blood 110, 4175–4178 (2007).
Perumbeti, A. & Malik, P. Therapy for β-globinopathies: a brief review and determinants for successful and safe correction. Ann. N. Y. Acad. Sci. 1202, 36–44 (2010).
Quek, L. & Thein, S. L. Molecular therapies in β-thalassaemia. Br. J. Haematol. 136, 353–365 (2007).
Boulad, F. et al. Safe mobilization of CD34+ cells in adults with β-thalassemia and validation of effective globin gene transfer for clinical investigation. Blood 123, 1483–1486 (2014).
Felfly, H. & Trudel, M. Successful correction of murine sickle cell disease with reduced stem cell requirements reinforced by fractionated marrow infusions. Br. J. Haematol. 148, 646–658 (2010).
Bartelink, I. H. et al. Association of busulfan exposure with survival and toxicity after haemopoietic cell transplantation in children and young adults: a multicentre, retrospective cohort analysis. Lancet Haematol. 3, e526–e536 (2016).
Hu, J. et al. Isolation and functional characterization of human erythroblasts at distinct stages: implications for understanding of normal and disordered erythropoiesis in vivo. Blood 121, 3246–3253 (2013).
Villa, E. et al. The E3 ligase UBR2 regulates cell death under caspase deficiency via Erk/MAPK pathway. Cell Death Dis. 11, 1041 (2020).
Berry, C. C., Ocwieja, K. E., Malani, N. & Bushman, F. D. Comparing DNA integration site clusters with scan statistics. Bioinformatics 30, 1493–1500 (2014).
Yoon, J. K. et al. HIV proviral DNA integration can drive T cell growth ex vivo. Proc. Natl Acad. Sci. USA 117, 32880–32882 (2020).
Katano, H. et al. Integration of HIV-1 caused STAT3-associated B cell lymphoma in an AIDS patient. Microbes Infect. 9, 1581–1589 (2007).
Bushman, F. D. Retroviral insertional mutagenesis in humans: evidence for four genetic mechanisms promoting expansion of cell clones. Mol. Ther. 28, 352–356 (2020).
Thompson, A. A. et al. Gene therapy in patients with transfusion-dependent β-thalassemia. N. Engl. J. Med. 378, 1479–1493 (2018).
Schneiderman, J. et al. Interim results from the phase 3 Hgb-207 (Northstar-2) and Hgb-212 (Northstar-3) studies of betibeglogene autotemcel gene therapy (LentiGlobin) for the treatment of transfusion-dependent β-thalassemia. Biol. Blood Marrow Transplant. 26, S87–S88 (2020).
Frangoul, H. et al. Safety and efficacy of CTX001 in patients with transfusion-dependent β-thalassemia and sickle cell disease: early results from the Climb THAL-111 and Climb SCD-121 studies of autologous CRISPR–CAS9-modified CD34+ hematopoietic stem and progenitor cells. Blood 136, 3–4 (2020).
Grochow, L. B. et al. Pharmacokinetics of busulfan: correlation with veno-occlusive disease in patients undergoing bone marrow transplantation. Cancer Chemother. Pharmacol. 25, 55–61 (1989).
Lawson, R., Staatz, C. E., Fraser, C. J. & Hennig, S. Review of the pharmacokinetics and pharmacodynamics of intravenous busulfan in paediatric patients. Clin. Pharmacokinet. 60, 17–51 (2021).
Strouse, C. et al. Risk score for the development of veno-occlusive disease after allogeneic hematopoietic cell transplant. Biol. Blood Marrow Transpl. 24, 2072–2080 (2018).
Marktel, S. et al. Platelet transfusion refractoriness in highly immunized β thalassemia children undergoing stem cell transplantation. Pediatr. Transpl. 14, 393–401 (2010).
Saito, A. M. et al. Lower costs associated with hematopoietic cell transplantation using reduced intensity vs high-dose regimens for hematological malignancy. Bone Marrow Transpl. 40, 209–217 (2007).
Svahn, B. M., Alvin, O., Ringden, O., Gardulf, A. & Remberger, M. Costs of allogeneic hematopoietic stem cell transplantation. Transplantation 82, 147–153 (2006).
La Nasa, G. et al. Long-term health-related quality of life evaluated more than 20 years after hematopoietic stem cell transplantation for thalassemia. Blood 122, 2262–2270 (2013).
Jones, R. J. & DeBaun, M. R. Leukemia after gene therapy for sickle cell disease: insertional mutagenesis, busulfan, both or neither. Blood 138, 942–947 (2021).
Hsieh, M. M. et al. Myelodysplastic syndrome unrelated to lentiviral vector in a patient treated with gene therapy for sickle cell disease. Blood Adv. 4, 2058–2063 (2020).
Li, Y. et al. Myeloid neoplasms in the setting of sickle cell disease: an intrinsic association with the underlying condition rather than a coincidence; report of 4 cases and review of the literature. Mod. Pathol. 32, 1712–1726 (2019).
Seminog, O. O., Ogunlaja, O. I., Yeates, D. & Goldacre, M. J. Risk of individual malignant neoplasms in patients with sickle cell disease: English national record linkage study. J. R. Soc. Med. 109, 303–309 (2016).
Brunson, A. et al. Increased risk of leukemia among sickle cell disease patients in California. Blood 130, 1597–1599 (2017).
Grimley, M. et al. Early results from a phase 1/2 study of Aru-1801 gene therapy for sickle cell disease (SCD): manufacturing process enhancements improve efficacy of a modified gamma globin lentivirus vector and reduced intensity conditioning transplant. Blood 136, 20–21 (2020).
Scaramuzza, S. et al. Clinical outcomes from a phase I/II gene therapy trial for patients affected by severe transfusion dependent β-thalassemia: two years follow up. Mol. Ther. 28, 169 (2020).
Marktel, S. et al. Intrabone hematopoietic stem cell gene therapy for adult and pediatric patients affected by transfusion-dependent β-thalassemia. Nat. Med. 25, 234–241 (2019).
Cavazzana-Calvo, M. et al. Transfusion independence and HMGA2 activation after gene therapy of human β-thalassaemia. Nature 467, 318–322 (2010).
Bank, A., Dorazio, R. & Leboulch, P. A phase I/II clinical trial of β-globin gene therapy for β-thalassemia. Ann. N. Y. Acad. Sci. 1054, 308–316 (2005).
Ikeda, K., Mason, P. J. & Bessler, M. 3′UTR-truncated Hmga2 cDNA causes MPN-like hematopoiesis by conferring a clonal growth advantage at the level of HSC in mice. Blood 117, 5860–5869 (2011).
Murakami, Y. et al. Deregulated expression of HMGA2 is implicated in clonal expansion of PIGA deficient cells in paroxysmal nocturnal haemoglobinuria. Br. J. Haematol. 156, 383–387 (2012).
Yu, K. R. et al. HMGA2 regulates the in vitro aging and proliferation of human umbilical cord blood-derived stromal cells through the mTOR/p70S6K signaling pathway. Stem Cell Res. 10, 156–165 (2013).
Bottardi, S., Ross, J., Pierre-Charles, N., Blank, V. & Milot, E. Lineage-specific activators affect β-globin locus chromatin in multipotent hematopoietic progenitors. EMBO J. 25, 3586–3595 (2006).
Jimenez, G., Griffiths, S. D., Ford, A. M., Greaves, M. F. & Enver, T. Activation of the β-globin locus control region precedes commitment to the erythroid lineage. Proc. Natl Acad. Sci. USA 89, 10618–10622 (1992).
Schumm, M. et al. Isolation of highly purified autologous and allogeneic peripheral CD34+ cells using the CliniMACS device. J. Hematother. 8, 209–218 (1999).
Alter, B. P., Rappeport, J. M., Huisman, T. H., Schroeder, W. A. & Nathan, D. G. Fetal erythropoiesis following bone marrow transplantation. Blood 48, 843–853 (1976).
Yang, S. et al. A simple and effective method to generate lentiviral vectors for ex vivo gene delivery to mature human peripheral blood lymphocytes. Hum. Gene Ther. Methods 23, 73–83 (2012).
Charrier, S. et al. Quantification of lentiviral vector copy numbers in individual hematopoietic colony-forming cells shows vector dose-dependent effects on the frequency and level of transduction. Gene Ther. 18, 479–487 (2011).
Shear, H. L. et al. Transgenic mice expressing human fetal globin are protected from malaria by a novel mechanism. Blood 92, 2520–2526 (1998).
Berry, C. C. et al. INSPIIRED: quantification and visualization tools for analyzing integration site distributions. Mol. Ther. Methods Clin. Dev. 4, 17–26 (2017).
Sherman, E. et al. INSPIIRED: a pipeline for quantitative analysis of sites of new DNA integration in cellular genomes. Mol. Ther. Methods Clin. Dev. 4, 39–49 (2017).
Berry, C. C. et al. Estimating abundances of retroviral insertion sites from DNA fragment length data. Bioinformatics 28, 755–762 (2012).
Acknowledgements
The authors would first like to thank the patients and families of those included in the trial and further acknowledge the expert care provided to patients by staff members of the Department of Pediatrics at Memorial Sloan Kettering Cancer Center. We thank R. Cristantielli, G. Gunset and E. Bechard for their further assistance in making our patients’ journeys and their follow-up pleasant and efficient. This clinical trial was supported by the Stavros Niarchos Foundation (to M.S.), the Memorial Hospital Research Fund (to F.B.), the Leonardo Giambrone Foundation (to M.S.) and the Cooley’s Anemia Foundation (to M.S.) for transduction, biosafety and clinical costs, and Errant Gene Therapy (to M.S.) for the TNS9.3.55 vector lot, produced at the Center for Biomedicine and Genetics in Duarte, California, and the MSKCC Support Grant (P30 CA008748). The funders had no role in study design, data collection and analysis, decision to publish or preparation of the manuscript. We also thank S. Avecilla, Q. He, C. Taylor, M. Fink, T. Wasielewska, S. Bartido, Y. Wang and the members of the Cell Therapy and Cell Engineering Facility who, for 7 years, have assisted in the manufacturing and monitoring of the CD34+ cell infusion products for our patients.
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F.B., A.M., P.M., S.A., S.P., S.R., R.B., R.D.M. and L.P. were involved in patient care. X.W., J.Q., K.T., F.D. and L.S. performed transductions and biosafety assays. F.K. and C.T. assembled and analyzed clinical data. J.M.S. and A.C. reviewed vector and sequence data. J.K.E., P.H., A.M.R., V.A.C., H.A., S.R. and F.D.B. conducted vector integration site analyses. J.K.E. and F.D.B. modeled and analyzed IS data. A.G. oversaw HPLC analysis. E.B. oversaw HPLC studies. N.M. oversaw bone marrow FACS analyses. F.B., A.M., I.R. and M.S. designed the study. F.B., A.M., F.K., F.D.B., I.R. and M.S. wrote the manuscript.
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The authors declare no competing interests. The TNS9.3.55 vector technology has been granted to Errant Gene Therapy without financial compensation.
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Extended data
Extended Data Fig. 1 Cell count recovery after conditioning and infusion of TNS9.3.55 transduced CD34 + HSPCs.
a, Platelet count (x109/l) after infusion. b, Absolute neutrophil count (x109/l) after infusion. Granulocyte colony stimulating factor (G-CSF) was administered for Patient 1 (day 13-16) and Patient 2 (day 16-18) during aplasia. Patient 1 received methylprednisolone intravenously for 3 consecutive days (day 16-18) for treating the engraftment syndrome.
Extended Data Fig. 2 β-globin expression in Patient 2 at 12 months post infusion.
a, HPLC chromatograms illustrating globin production of erythroid cells from four individual BFU-Es derived from bone marrow obtained from Patient 2 at 12 months post infusion. Top chromatograms: two representative examples from two individual, non-transduced BFU-E from Patient 2; lower chromatograms: two representative examples from two individual, transduced BFU-E from Patient 2. b, β-globin to α-globin ration in erythroids derived from untransduced and transduced HSCs obtained from Patient 2 at 12 months post infusion. The β/α expression ratio determined by HPLC in single BFU-Es increased from a mean of 0.11 to 0.38 in BFU-Es harboring a single copy of the integrated vector, representing a gain of 0.27. Results from BFU-Es derived from untransduced HSCs (n = 2; VCN = 0) and from BFU-Es derived from transduced HSCs (n = 4; VCN = 1). All data are mean ± s.e.m.
Extended Data Fig. 3 Erythropoietic maturation in bone marrow (Patient 2).
Terminal erythroid differentiation begins with proerythroblasts differentiating into basophilic, then polychromatic, then orthochromatic erythroblasts that enucleate to become reticulocytes. Each distinct stage of terminal human erythroid differentiation can be distinguished using a combination of cell surface markers for glycophorine A (GPA), band 3 and α4 integrin. a, Representative flow cytometry plot of band 3 vs α4-integrin of GPA + cells in normal erythropoiesis: proerythroblasts (I), early basophilic (II), late basophilic (III), polychromatic (IV), and orthochromatic erythroblasts (V) and reticulocytes (VI). The box plot represents the quantitation of the proportion of cells at each distinct stage of maturation after normalization based on total nucleated erythroid cells (I-V) as 100% as described in ref. 29. The left panel is adapted from ref. 29. b, Bone marrow erythroblasts from Patient 2 were analyzed by flow cytometry at 6, 12 and 24 months post infusion stained with GPA, α4-integrin, and band 3. Plot of band 3 vs α4-integrin of GPA + cells represents the quantitation of distinct stages of maturation of erythroblasts as described in a. c, Quantitation of the proportion of cells at each distinct stage of maturation after normalization to total nucleated erythroid cells (I-V).
Extended Data Fig. 4 Engraftment of transduced cells in bone marrow.
Vector copy number (VCN) in erythroid glycophorine A + (GPA + ) cells and CD45 + cells sorted from bone marrow of patients.
Extended Data Fig. 5 Gini and Shannon Index values.
Timepoints in months.
Extended Data Fig. 6 Annotation of the genes STAT3 and STAT5A on chromosome 17.
Illustration of a cluster of transgene integrations in the first intron of STAT3. Six integration sites were detected at year six, zero were detected pre-transplant. Four out of six integration sites detected were in the same transcriptional orientation as STAT3.
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Boulad, F., Maggio, A., Wang, X. et al. Lentiviral globin gene therapy with reduced-intensity conditioning in adults with β-thalassemia: a phase 1 trial. Nat Med 28, 63–70 (2022). https://doi.org/10.1038/s41591-021-01554-9
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DOI: https://doi.org/10.1038/s41591-021-01554-9
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