Subjects

Abstract

ß-thalassemia is caused by ß-globin gene mutations resulting in reduced (β+) or absent (β0) hemoglobin production. Patient life expectancy has recently increased, but the need for chronic transfusions in transfusion-dependent thalassemia (TDT) and iron chelation impairs quality of life1. Allogeneic hematopoietic stem cell (HSC) transplantation represents the curative treatment, with thalassemia-free survival exceeding 80%. However, it is available to a minority of patients and is associated with morbidity, rejection and graft-versus-host disease2. Gene therapy with autologous HSCs modified to express ß-globin represents a potential therapeutic option. We treated three adults and six children with ß0 or severe ß+ mutations in a phase 1/2 trial (NCT02453477) with an intrabone administration of HSCs transduced with the lentiviral vector GLOBE. Rapid hematopoietic recovery with polyclonal multilineage engraftment of vector-marked cells was achieved, with a median of 37.5% (range 12.6–76.4%) in hematopoietic progenitors and a vector copy number per cell (VCN) of 0.58 (range 0.10–1.97) in erythroid precursors at 1 year, in absence of clonal dominance. Transfusion requirement was reduced in the adults. Three out of four evaluable pediatric participants discontinued transfusions after gene therapy and were transfusion independent at the last follow-up. Younger age and persistence of higher VCN in the repopulating hematopoietic cells are associated with better outcome.

Access optionsAccess options

Rent or Buy article

Get time limited or full article access on ReadCube.

from$8.99

All prices are NET prices.

Code availability

Software scripts developed for the integration site analysis will be made available upon request from the corresponding author.

Data availability

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 the paper were generated as part of clinical trials and may be subject to patient confidentiality. Any data and materials that can be shared will be released via a Material Transfer Agreement.

Additional information

Publisher’s note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

References

  1. 1.

    Taher, A. T., Weatherall, D. J. & Cappellini, M. D. Thalassaemia. Lancet 391, 155–167 (2018).

  2. 2.

    Baronciani, D. et al. Hemopoietic stem cell transplantation in thalassemia: a report from the European Society for Blood and Bone Marrow Transplantation Hemoglobinopathy Registry, 2000-2010. Bone. Marrow. Transplant. 51, 536–541 (2016).

  3. 3.

    Naldini, L. Ex vivo gene transfer and correction for cell-based therapies. Nat. Rev. Genet. 12, 301–315 (2011).

  4. 4.

    Modell, B. & Darlison, M. Global epidemiology of haemoglobin disorders and derived service indicators. Bull. World Health Organ. 86, 480–487 (2008).

  5. 5.

    Cavazzana-Calvo, M. et al. Transfusion independence and HMGA2 activation after gene therapy of human beta-thalassaemia. Nature 467, 318–322 (2010).

  6. 6.

    Thompson, A. et al. Gene therapy in patients with transfusion-dependent β-thalassemia. N. Engl. J. Med. 378, 1479–1493 (2018).

  7. 7.

    Roselli, E. A. et al. Correction of beta-thalassemia major by gene transfer in haematopoietic progenitors of pediatric patients. EMBO Mol. Med. 2, 315–328 (2010).

  8. 8.

    Miccio, A. et al. In vivo selection of genetically modified erythroblastic progenitors leads to long-term correction of beta-thalassemia. Proc. Natl Acad. Sci. USA 105, 10547–10552 (2008).

  9. 9.

    Miccio, A. et al. The GATA1-HS2 enhancer allows persistent and position-independent expression of a beta-globin transgene. PLoS ONE 6, e27955 (2011).

  10. 10.

    Lidonnici, M. R. et al. Multiple integrated non-clinical studies predict safety of lentiviral mediated gene therapy for beta thalassemia. Mol. Ther. Methods Clin. Dev. 13, 9–28 (2018).

  11. 11.

    Frassoni, F. et al. Direct intrabone transplant of unrelated cord-blood cells in acute leukaemia: a phase I/II study. Lancet. Oncol. 9, 831–839 (2008).

  12. 12.

    Okada, M. et al. A prospective multicenter phase II study of intrabone marrow transplantation of unwashed cord blood using reduced-intensity conditioning. Eur. J. Haematol. 100, 335–343 (2018).

  13. 13.

    Murata, M. et al. Phase II study of intrabone single unit cord blood transplantation for hematological malignancies. Cancer Sci. 108, 1634–1639 (2017).

  14. 14.

    Rocha, V. et al. Unrelated cord blood transplantation: outcomes after single-unit intrabone injection compared with double-unit intravenous injection in patients with hematological malignancies. Transplantation 95, 1284–1291 (2013).

  15. 15.

    Bonifazi, F., et al. Intrabone transplant provides full stemness of cord blood stem cells with fast hematopoietic recovery and low GVHD rate: results from a prospective study. Bone Marrow Transplant. https://doi.org/10.1038/s41409-018-0335-x (2018).

  16. 16.

    Lidonnici, M. R. 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 102, e120–e124 (2017).

  17. 17.

    Yahata, T. et al. A highly sensitive strategy for SCID-repopulating cell assay by direct injection of primitive human hematopoietic cells into NOD/SCID mice bone marrow. Blood 101, 2905–2913 (2003).

  18. 18.

    Feng, Q. et al. Nonhuman primate allogeneic hematopoietic stem cell transplantation by intraosseus vs intravenous injection: engraftment, donor cell distribution, and mechanistic basis. Exp. Hematol. 36, 1556–1566 (2008).

  19. 19.

    Chiesa, R., S, J., Winter, R., Prunty, H., Nademi, Z., Slatter, M. & Veys, P. Phase II trial to define the therapeutic index of treosulfan for myeloablative conditioning in haematopoietic stem cell transplant. BMJ 102, A23 (2017).

  20. 20.

    Mansilla-Soto, J., Riviere, I., Boulad, F. & Sadelain, M. Cell and gene therapy for the beta-thalassemias: advances and prospects. Hum. Gene Ther. 27, 295–304 (2016).

  21. 21.

    Remberger, M. et al. Toxicological effects of fludarabine and treosulfan conditioning before allogeneic stem-cell transplantation. Int. J. Hematol. 106, 471–475 (2017).

  22. 22.

    Wiebking, V. et al. Reduced toxicity, myeloablative HLA-haploidentical hematopoietic stem cell transplantation with post-transplantation cyclophosphamide for sickle cell disease. Ann. Hematol. 96, 1373–1377 (2017).

  23. 23.

    Mathews, V. et al. Improved clinical outcomes of high risk beta thalassemia major patients undergoing a HLA matched related allogeneic stem cell transplant with a treosulfan based conditioning regimen and peripheral blood stem cell grafts. PLoS ONE 8, e61637 (2013).

  24. 24.

    Sessa, M. et al. Lentiviral haemopoietic stem-cell gene therapy in early-onset metachromatic leukodystrophy: an ad-hoc analysis of a non-randomised, open-label, phase 1/2 trial. Lancet 388, 476–487 (2016).

  25. 25.

    Ribeil, J. A. et al. Gene therapy in a patient with sickle cell disease. N. Engl. J. Med. 376, 848–855 (2017).

  26. 26.

    Eichler, F. et al. DA. Hematopoietic stem-cell gene therapy for cerebral adrenoleukodystrophy. N. Engl. J. Med. 377, 1630–1638 (2017).

  27. 27.

    Marktel, S. et al. Platelet transfusion refractoriness in highly immunized beta thalassemia children undergoing stem cell transplantation. Pediatr. Transplant. 14, 393–401 (2010).

  28. 28.

    Biffi, A. et al. Lentiviral hematopoietic stem cell gene therapy benefits metachromatic leukodystrophy. Science 341, 1233158 (2013).

  29. 29.

    Andreani, M. et al. Quantitatively different red cell/nucleated cell chimerism in patients with long-term, persistent hematopoietic mixed chimerism after bone marrow transplantation for thalassemia major or sickle cell disease. Haematologica 96, 128–133 (2011).

  30. 30.

    Aiuti, A. et al. Lentiviral hematopoietic stem cell gene therapy in patients with Wiskott–Aldrich syndrome. Science 341, 1233151 (2013).

  31. 31.

    Cartier, N. et al. Hematopoietic stem cell gene therapy with a lentiviral vector in X-linked adrenoleukodystrophy. Science 326, 818–823 (2009).

  32. 32.

    de Ridder, J., Uren, A., Kool, J., Reinders, M. & Wessels, L. Detecting statistically significant common insertion sites in retroviral insertional mutagenesis screens. PLoS Comput. Biol. 2, e166 (2006).

  33. 33.

    Wood, J. C. et al. MRI R2 and R2* mapping accurately estimates hepatic iron concentration in transfusion-dependent thalassemia and sickle cell disease patients. Blood 106, 1460–1465 (2005).

  34. 34.

    Pantin, J. M. et al. Optimization of intrabone delivery of hematopoietic progenitor cells in a swine model using cell radiolabeling with [89]zirconium. Am. J. Transplant. 15, 606–617 (2015).

  35. 35.

    Dalle, J. H. et al. State-of-the-art fertility preservation in children and adolescents undergoing haematopoietic stem cell transplantation: a report on the expert meeting of the Paediatric Diseases Working Party (PDWP) of the European Society for Blood and Marrow Transplantation (EBMT) in Baden, Austria, 29-30 September 2015. Bone. Marrow. Transplant. 52, 1029–1035 (2017).

  36. 36.

    Hu, J. 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).

Download references

Acknowledgements

The clinical trial was supported initially by Fondazione Telethon and by GlaxoSmithKline (GSK). We wish to acknowledge Fondazione Telethon for continuous support and strategic guidance. GSK has licensed the thalassemia HSC gene therapy in August 2017 and transferred to Orchard Therapeutics in April 2018. We thank the patients and families who are participating in this study; The Association of Patients with Thalassaemia and Drepanocytosis in Lombardy, Italy, for facilitating communication between patients, families and health care operators. We thank all physicians that have referred patients for participation, the clinical and laboratory staff of the Ospedale San Raffaele Stem Cell Program for patient care and data collection, the Paediatric Clinical Research Unit, S. Zancan and the personnel of SR-TIGET Clinical Trial Office for regulatory support. We thank A. Spinelli of the Ospedale San Raffaele Experimental Imaging Centre for bioluminescence acquisition and analysis. We are grateful to H. Prunty and R. Chiesa for supervision and interpretation of the treosulfan pharmacokinetics analysis (Great Ormond Street Hospital, London, UK).

Author information

Author notes

  1. These authors contributed equally: Sarah Marktel, Samantha Scaramuzza, Fabio Ciceri, Alessandro Aiuti and Giuliana Ferrari.

Affiliations

  1. San Raffaele Telethon Institute for Gene Therapy (SR-TIGET), IRCCS San Raffaele Scientific Institute, Milan, Italy

    • Sarah Marktel
    • , Samantha Scaramuzza
    • , Maria Pia Cicalese
    • , Maria Rosa Lidonnici
    • , Valeria Calbi
    • , Maria Ester Bernardo
    • , Claudia Rossi
    • , Andrea Calabria
    • , Fabrizio Benedicenti
    • , Giulio Spinozzi
    • , Annamaria Aprile
    • , Alessandra Bergami
    • , Miriam Casiraghi
    • , Giulia Consiglieri
    • , Michela Gabaldo
    • , Eugenio Montini
    • , Luigi Naldini
    • , Fabio Ciceri
    • , Alessandro Aiuti
    •  & Giuliana Ferrari
  2. Haematology and BMT Unit, IRCCS San Raffaele Scientific Institute, Milan, Italy

    • Sarah Marktel
    • , Fabio Giglio
    • , Andrea Assanelli
    •  & Fabio Ciceri
  3. Pediatric Immunohematology, IRCCS San Raffaele Scientific Institute, Milan, Italy

    • Maria Pia Cicalese
    • , Valeria Calbi
    • , Maria Ester Bernardo
    • , Miriam Casiraghi
    • , Giulia Consiglieri
    •  & Alessandro Aiuti
  4. Center of Biostatistics for Clinical Epidemiology, School of Medicine and Surgery, University of Milan-Bicocca, Milan, Italy

    • Stefania Galimberti
    •  & Maria Grazia Valsecchi
  5. Blood Transfusion Service, IRCCS San Raffaele Scientific Institute, Milan, Italy

    • Raffaella Milani
    • , Salvatore Gattillo
    • , Milena Coppola
    •  & Luca Santoleri
  6. Pediatric Department University of Milano-Bicocca, San Gerardo Hospital, Monza, Italy

    • Nicoletta Masera
  7. Pediatric Clinic/DH, Fondazione IRCCS Ca’ Granda, Milan, Italy

    • Emanuela D’Angelo
    •  & Nadia Mirra
  8. Ospedale Pediatrico Microcitemico “A.Cao”, A.O. “G.Brotzu”, Cagliari, Italy

    • Raffaella Origa
  9. Department of Woman, Child and General and Specialist Surgery, Università degli studi della Campania “Luigi Vanvitelli”, Napoli, Italy

    • Immacolata Tartaglione
    •  & Silverio Perrotta
  10. Department of Chemical Pathology, Great Ormond Street Hospital NHS Foundation Trust, London, UK

    • Robert Winter
  11. Immunohematology and Transfusion Medicine Service, Fondazione IRCCS Policlinico S. Matteo, Pavia, Italy

    • Gianluca Viarengo
  12. Rare Disease Center, Fondazione IRCCS Ca’ Granda, University of Milan, Milan, Italy

    • Giovanna Graziadei
    •  & Maria Domenica Cappellini
  13. Vita-Salute San Raffaele University, Milan, Italy

    • Luigi Naldini
    • , Fabio Ciceri
    • , Alessandro Aiuti
    •  & Giuliana Ferrari

Authors

  1. Search for Sarah Marktel in:

  2. Search for Samantha Scaramuzza in:

  3. Search for Maria Pia Cicalese in:

  4. Search for Fabio Giglio in:

  5. Search for Stefania Galimberti in:

  6. Search for Maria Rosa Lidonnici in:

  7. Search for Valeria Calbi in:

  8. Search for Andrea Assanelli in:

  9. Search for Maria Ester Bernardo in:

  10. Search for Claudia Rossi in:

  11. Search for Andrea Calabria in:

  12. Search for Raffaella Milani in:

  13. Search for Salvatore Gattillo in:

  14. Search for Fabrizio Benedicenti in:

  15. Search for Giulio Spinozzi in:

  16. Search for Annamaria Aprile in:

  17. Search for Alessandra Bergami in:

  18. Search for Miriam Casiraghi in:

  19. Search for Giulia Consiglieri in:

  20. Search for Nicoletta Masera in:

  21. Search for Emanuela D’Angelo in:

  22. Search for Nadia Mirra in:

  23. Search for Raffaella Origa in:

  24. Search for Immacolata Tartaglione in:

  25. Search for Silverio Perrotta in:

  26. Search for Robert Winter in:

  27. Search for Milena Coppola in:

  28. Search for Gianluca Viarengo in:

  29. Search for Luca Santoleri in:

  30. Search for Giovanna Graziadei in:

  31. Search for Michela Gabaldo in:

  32. Search for Maria Grazia Valsecchi in:

  33. Search for Eugenio Montini in:

  34. Search for Luigi Naldini in:

  35. Search for Maria Domenica Cappellini in:

  36. Search for Fabio Ciceri in:

  37. Search for Alessandro Aiuti in:

  38. Search for Giuliana Ferrari in:

Contributions

S.M., M.P.C., F.G. and V.C. contributed to the study design, patient follow-up, data collection, interpretation and manuscript writing. S.S. contributed to laboratory experiments, molecular analysis, data collection, interpretation and manuscript writing. A. Assanelli, M.E.B., M.C., G.C., N. Masera, E.A., N. Mirra, R.O., I.T., S.P., G.G. contributed to patient follow-up, data collection and interpretation. A.B. and M.G. contributed to data collection and regulatory applications. M.R.L. designed and performed the studies of the experimental model of intrabone HSC injection and contributed to laboratory experiments and data interpretation. C.R. and A. Aprile contributed to laboratory experiments and data interpretation. A.C., F.B., G.S. and E.M. contributed to integration sites sequencing, mapping and analysis. R.M., S. Gattillo, M.C., G.V. and L.S. were responsible for stem cell collection, characterization and data interpretation. R.W. was responsible for treosulfan pharmacokinetics data. S. Galimberti and M.G.V. contributed to study design, data interpretation and were responsible of statistical analysis. L.N., M.D.C., F.C. and A. Aiuti contributed to study design, data interpretation and manuscript writing. G.F. contributed to study design, supervision of molecular studies and laboratory experiments, data interpretation and manuscript writing.

Competing interests

The San Raffaele Telethon Institute for Gene Therapy (SR-TIGET) is a joint venture between the Telethon Foundation and Ospedale San Raffaele. The Telethon Foundation and Ospedale San Raffaele are entitled to receive milestone payments and royalties on commercialization of this therapy. A. Aiuti is the principal investigator of the SR-TIGET clinical trial of gene therapy for ß-thalassemia and S.M., M.P.C., F.G., V.C., M.D.C. and F.C. are co-investigators or sub-investigators. G.F. is the scientific director of the study. L.N. is an inventor on patents on lentiviral vector technology filed by the Salk Institute, Cell Genesys, Telethon Foundation and/or Ospedale San Raffaele, and may be entitled to receive some financial benefits from the licensing of such patents. All authors declare no other competing interests.

Corresponding author

Correspondence to Giuliana Ferrari.

Extended data

  1. Extended Data Fig. 1 In vivo distribution of marked Lin- cells in different districts of the mouse body (intravenous versus intrabone).

    HSPCs were injected in mice by intravenous or intrabone administration. Bioluminescence imaging analysis was used to evaluate the distribution of HSPCs expressing luciferase in the different body districts at the indicated time points. Representative images of three animals for each group.

  2. Extended Data Fig. 2 Analysis of biodistribution of transduced HSCs (intravenous versus intrabone).

    a, Mice were analyzed using bioluminescence imaging to evaluate the dynamics of transduced of hematopoietic stem and progenitor cells (HSPCs) in the different districts of the animals at the indicated time points. Total flux was measured in the different districts of the animal body. At earlier time points, the levels of intravenous transplanted cells in the lung (3 h  P = 0.0120, 24 h P = 0.0216) and in liver-spleen (3 h P = 0.0042, 24 h P = 0.0014 and 48 h P = 0.0440) were significantly higher than those of intrabone injected cells. (see Extended Data Fig. 2) b, Evaluation of hematological parameters (neutrophil, left panel and platelet count, right panel) in mice transplanted with HSPCs by intrabone (N = 6) or intravenous (N = 6) (P = 0.0172), a two-tailed t-test was applied. (see Extended Data Suppl. Fig. 2). c, Evaluation of hematopoietic reconstitution of HSPCs transplanted by intrabone or intravenous. Engraftment was analyzed by flow cytometry at the different time points in the peripheral blood (2–4–7 months) (see Extended Data Fig. 2). Engraftment was calculated as %CD45.1pos cells/(CD45.1pos + CD45.2pos) cells: 2 months (P = 0.0246) and 4 months (P = 0.0195). A two-tailed t-test was applied. Source Data

  3. Extended Data Fig. 3 Structure of the GLOBE lentiviral vector.

    Drug product was manufactured using clinical-grade GLOBE lentiviral vector lots. LTR, Long Terminal Repeat; CMV, enhancer/promoter region of cytomegalovirus; RRE, rev-responsive element; SD, splicing donor site; SA, splicing acceptor site; cPPT, central polypurine tract; βp, globin promoter; HS, DNase I-hypersensitive sites; LCR, Locus Control Region.

  4. Extended Data Fig. 4 Basal level of expression in adult participants.

    Levels of residual hemoglobin B expression measured by qPCR at screening in erythroid cultured cells from adult participants, expressed as a ratio on the housekeeping gene GAPDH. Results of patients are expressed as a mean of two replicates.

  5. Extended Data Fig. 5 Terminal erythropoiesis in transfusion independent participant 4.

    a, Gating strategy (left panel) and erythroid maturation (right panel) measured by flow-cytometry analysis on bone marrow isolated from participant 4 at 1 yr post-gene-therapy. Erythroid cells at different maturation stages are reported as percentage of total gated cells. Pro: proerythroblast; baso: basophilic erythroblast; poly: polychromatophilic erythroblast; ortho: orthochromatic erythroblast. In healthy donor, the physiologic progression of normal human terminal erythroid differentiation follows a ratio of Pro/Early Baso to Late Baso/Poly/Ortho, of 1/2/4/8/16 (as from Hu et al.36) b, Peripheral blood smear from Pt4 at 1 year follow-up. May–Grunwald–Giemsa staining, magnification ×1,000.

  6. Extended Data Fig. 6 Clonal abundance over time.

    For each participant, tissue and population analyzed (whole peripheral blood and whole bone marrow), the clonal abundance over time is represented (x axis, days after gene therapy) with a stacked bar plot in which each clone is a different color with the height in relative proportion with the number of retrieved cells (percentage); ribbons connect tracked clones between two consecutive time points. Clonal abundance of peripheral blood for participants 1 and 4 are shown in Fig. 3 of the main text. For participants 8 and 9 data are not available yet.

  7. Extended Data Fig. 7 Correlation between transduction efficiency and the clonal population diversity index through integration site analysis.

    a, Correlation between transduction efficiency in cells from bone marrow samples for all patients and all time points and the clonal population diversity index through integration site analysis. b, Clonality indexes obtained from whole bone marrow and peripheral blood cells of adults and pediatric subjects. Boxplots shows distributions of data (dots) in quartiles, where in each boxplot the upper/lower whiskers refer to 75th/25th percentile, the box refers to inter-quantile range and the median is shown with the solid line. A non-parametric exact Wilcoxon test was used to compare intregration site diversity between groups of patients (adults versus pediatrics) and a paired version was adopted to compare integration site diversity between tissues (bone marrow versus peripheral blood) (two-sided tests with alpha = 0.05).

  8. Extended Data Fig. 8 Clonal abundance over time in erythroid bone marrow compartment.

    a,b, Glycophorin A+ (a) and CD36+ (b) samples. Plots exploit a stacked bar plot representation (x axis, months post-gene-therapy; y axis, relative proportion of clonal abundance, number of cells per clone) in which each color is associated to a distinct clone, tracked with a ribbon between two consecutive time points. For participants 8 and 9 data are not available yet.

  9. Extended Data Fig. 9 Genomic profile of vector integration sites.

    Genomic profile of vector integration sites. a, Vector distribution profile in all chromosomes in comparison with that of other lentiviral vector trials participants. For participants 8 and 9 data are not available yet. b, Distribution of IS surrounding genes, centered on TSS for participants 1, 4 and 7. c, Gene ontology for participant 1 for the classes biological processes, cellular components, and molecular functions.

Supplementary information

  1. Supplementary Information

    Supplementary Tables 1–5, Supplementary Methods and Supplementary Results

  2. Reporting Summary

Source data

  1. Source Data Extended Data Fig. 2

    Statistical Source Data

About this article

Publication history

Received

Accepted

Published

DOI

https://doi.org/10.1038/s41591-018-0301-6