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Intrabone hematopoietic stem cell gene therapy for adult and pediatric patients affected by transfusion-dependent ß-thalassemia



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.

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

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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

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.

Correspondence to Giuliana Ferrari.

Extended data

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.

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

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.

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.

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.

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.

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).

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.

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.

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Fig. 1: Engraftment of transduced cells in peripheral blood and bone marrow of adult and pediatric participants.
Fig. 2: Clinical outcome.
Fig. 3: Integration site analysis.
Extended Data Fig. 1: In vivo distribution of marked Lin- cells in different districts of the mouse body (intravenous versus intrabone).
Extended Data Fig. 2: Analysis of biodistribution of transduced HSCs (intravenous versus intrabone).
Extended Data Fig. 3: Structure of the GLOBE lentiviral vector.
Extended Data Fig. 4: Basal level of expression in adult participants.
Extended Data Fig. 5: Terminal erythropoiesis in transfusion independent participant 4.
Extended Data Fig. 6: Clonal abundance over time.
Extended Data Fig. 7: Correlation between transduction efficiency and the clonal population diversity index through integration site analysis.
Extended Data Fig. 8: Clonal abundance over time in erythroid bone marrow compartment.
Extended Data Fig. 9: Genomic profile of vector integration sites.