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Dynamics of genetically engineered hematopoietic stem and progenitor cells after autologous transplantation in humans

Abstract

Hematopoietic stem and progenitor cells (HSPC) are endowed with the role of generating and maintaining lifelong the extremely diverse pool of blood cells1. Clinically, transplantation of human HSPC from an allogeneic healthy donor or infusion of autologous gene-corrected HSPC can effectively replenish defective blood cell production caused by congenital or acquired disorders2,3,4,5,6,7,8,9. However, due to methodological and ethical constraints that have limited the study of human HSPC primarily to in vitro assays10 or xenotransplantation models11,12, the in vivo activity of HSPC has to date remained relatively unexplored in humans13,14,15,16. Here we report a comprehensive study of the frequencies, dynamics and output of seven HSPC subtypes in humans that was performed by tracking 148,093 individual clones in six patients treated with lentiviral gene therapy using autologous HSPC transplantation and followed for up to 5 years. We discovered that primitive multipotent progenitor and hematopoietic stem cell (HSC) populations have distinct roles during the initial reconstitution after transplant, compared with subsequent steady-state phases. Furthermore, we showed that a fraction of in vitro–activated HSC are resilient and undergo a defined delayed activation period upon transplant. Finally, our data support the concept that early lymphoid-biased progenitors might be capable of long-term survival, such that they can be maintained independently of their continuous production from HSC. Overall, this study provides comprehensive data on HSPC dynamics after autologous transplantation and gene therapy in humans.

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Fig. 1: Phenotypic characterization and quantification of HSPC subsets in WAS patients after GT.
Fig. 2: VCN evaluation and hierarchical modeling of human HSPC subsets.
Fig. 3: Analysis of the distribution of IS in myeloid and lymphoid lineages, as assessed in HSPC subpopulations, BM progenitors and PB cell populations.
Fig. 4: Qualitative assessment of HSC versus MPP latency and a proposed model of hematopoietic reconstitution.

Data availability

All data generated or analyzed during this study are included in this published article (and its supplementary information files). Additional raw data files will be made available by the corresponding authors upon reasonable request.

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Acknowledgements

This work was supported by Fondazione Telethon (TIGET Core Grant B2, A.A.), the Italian Ministero della Salute (Programma di Rete, NET-2011-02350069, A.A.) and the European Commission (ERARE-3-JTC 2015 EUROCID, A.A.). The work of D.P. and L.B. on bioinformatics and statistical analyses of the results and on manuscript preparation phases was performed utilizing the resources of the Gene Therapy Program of the Dana Farber/Boston Children’s Cancer and Blood Disorders Center. We thank F. Ciceri, M. G. Roncarolo and all medical and nursing staff of the Pediatric Immunohematology and Bone Marrow Transplantation Unit of the San Raffaele Scientific Institute and the San Raffaele Stem Cell Programme; S. Zancan, M. Casiraghi, S. Darin, M. Facchini, G. Tomaselli and all TIGET Clinical Trial Office personnel for clinical trial management and support; the GlaxoSmithKline research and development team for revising the manuscript; M. Gabaldo for support with project management; and P. Massariello, G. Vallanti, M. Manfredini and other MolMed staff for patient cell manipulation. We thank A. Ditadi for critically reviewing the manuscript. We thank C. Villa, E. Canonico and M. Romanò of the Flow Cytometry Resource, Advanced Cytometry Technical Applications Laboratory (FRACTAL) at Ospedale San Raffaele for cell sorting and technical help with instrumentation. We are indebted to the patients and their families for their commitment.

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S.S., L.B.-R., F.D. and D.P. are coauthors of this work. S.S. performed phenotypic characterization, IS retrieval of HSPC progenitors and additional molecular testing for VCN estimation, interpreted the data and wrote the manuscript; L.B.-R. performed phenotypic characterization and isolation of HSPC subpopulations, interpreted the data and wrote the manuscript; F.D. performed LAM–PCR and VCN estimation for all BM and PB patient samples; D.P. mapped IS, designed and applied mathematical models of hematopoietic hierarchy and analyzed lymphoid/myeloid contribution of subsets isolated from GT patients; F.A.S. and S.G. performed isolation of patient cell lineages; L.L. generated the bioinformatics pipeline for IS mapping; M.P.C. and F.F. provided WAS patients’ BM and PB samples and clinical data; A.A. contributed as PI by interpreting data, supervising the project and revising the manuscript; L.B. designed IS analyses, supervised the project and wrote the manuscript.

Corresponding authors

Correspondence to Alessandro Aiuti or Luca Biasco.

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Competing interests

The WAS gene therapy trial (NCT01515462) was originally sponsored by Fondazione Telethon and promoted by San Raffaele Telethon Institute for Gene Therapy (SR-TIGET). GlaxoSmithKline subsequently in-licensed the investigational product (GSK2696275) and became the financial and regulatory sponsor of the study. In April 2018 the license was transferred to Orchard Therapeutics. A.A. is the principal investigator (PI) of the clinical trial. L.B. is a consultant to GlaxoSmithKline for the assessment of safety of the WAS GT. All the other authors declare no competing interests. Readers are welcome to comment on the online version of the paper.

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Supplementary information

Supplementary Text and Figures

Supplementary Figures 1–10, Supplementary Tables 1–6 and Supplementary Methods

Reporting Summary

Supplementary Dataset 1

Recapture probabilities of IS

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Scala, S., Basso-Ricci, L., Dionisio, F. et al. Dynamics of genetically engineered hematopoietic stem and progenitor cells after autologous transplantation in humans. Nat Med 24, 1683–1690 (2018). https://doi.org/10.1038/s41591-018-0195-3

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