Abstract

The β-haemoglobinopathies, such as sickle cell disease and β-thalassaemia, are caused by mutations in the β-globin (HBB) gene and affect millions of people worldwide. Ex vivo gene correction in patient-derived haematopoietic stem cells followed by autologous transplantation could be used to cure β-haemoglobinopathies. Here we present a CRISPR/Cas9 gene-editing system that combines Cas9 ribonucleoproteins and adeno-associated viral vector delivery of a homologous donor to achieve homologous recombination at the HBB gene in haematopoietic stem cells. Notably, we devise an enrichment model to purify a population of haematopoietic stem and progenitor cells with more than 90% targeted integration. We also show efficient correction of the Glu6Val mutation responsible for sickle cell disease by using patient-derived stem and progenitor cells that, after differentiation into erythrocytes, express adult β-globin (HbA) messenger RNA, which confirms intact transcriptional regulation of edited HBB alleles. Collectively, these preclinical studies outline a CRISPR-based methodology for targeting haematopoietic stem cells by homologous recombination at the HBB locus to advance the development of next-generation therapies for β-haemoglobinopathies.

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Acknowledgements

D.P.D. was supported by a Stanford Child Health Research Institute (CHRI) Grant and Postdoctoral Award. R.O.B. was supported by an Individual Postdoctoral grant (DFF–1333-00106B) and a Sapere Aude, Research Talent grant (DFF–1331-00735B), both from the Danish Council for Independent Research, Medical Sciences. M.H.P. acknowledges the support of the Amon Carter Foundation, the Laurie Kraus Lacob Faculty Scholar Award in Pediatric Translational Research and NIH grant support PN2EY018244, R01-AI097320 and R01-AI120766. We thank D. Russell for the pDGM6 plasmid, H.-P. Kiem for scAAV6, G. de Alencastro and M. Kay for help with AAV production, the Binns Program for Cord Blood Research at Stanford University for cord-blood-derived CD34+ HSPCs. We also thank Lonza (A. Toell and G. Alberts) for donating the LV unit for performing large-scale genome-editing studies. We further thank members of the Porteus laboratory, D. DiGiusto and M. G. Roncarolo for input, comments and discussion.

Author information

Author notes

    • Daniel P. Dever
    •  & Rasmus O. Bak

    These authors contributed equally to this work.

    • Nobuko Uchida

    Present address: Institute of Stem Cell Biology and Regenerative Medicine, Stanford University, Stanford, California 94305, USA.

Affiliations

  1. Department of Pediatrics, Stanford University, Stanford, California 94305, USA

    • Daniel P. Dever
    • , Rasmus O. Bak
    • , Joab Camarena
    • , Gabriel Washington
    • , Carmencita E. Nicolas
    • , Mara Pavel-Dinu
    • , Nivi Saxena
    • , Alec B. Wilkens
    • , Sruthi Mantri
    • , Ayal Hendel
    • , Kenneth I. Weinberg
    •  & Matthew H. Porteus
  2. Department of Medicine, Division of Hematology, Cancer Institute, and Institute for Stem Cell Biology and Regenerative Medicine, Stanford University, Stanford, California 94305, USA

    • Andreas Reinisch
    •  & Ravindra Majeti
  3. Stem Cells, Inc. 7707 Gateway Blvd., Suite 140, Newark, California 94560, USA

    • Nobuko Uchida
  4. Division of Hematology/Oncology, Department of Pediatrics, Stanford University School of Medicine, Stanford, California 94035, USA

    • Anupama Narla

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Contributions

D.P.D. and R.O.B. contributed equally to this work as well as performed and designed most of the experiments. D.P.D., R.O.B., A.R. and C.E.N. designed, executed and analysed engraftment studies. J.C. performed some HBB homologous recombination, NHEJ and tNGFR experiments. G.W. performed in vitro erythrocyte differentiation experiments. M.P.-D. assisted with large-scale electroporation experiments. N.S. ran and analysed HSPC immunophenotyping experiments. A.B.W. scored and collected methylcellulose clones. N.U. sorted freshly isolated HSCs before targeting. S.M. purified CD34+ HSPCs from peripheral blood of patients with SCD. A.N. was the attending physician who oversaw transfusions of patients with SCD. A.H., R.M. and K.I.W. contributed to experimental design and data interpretation. M.H.P directed the research and participated in the design and interpretation of the experiments and the writing of the manuscript. D.P.D. and R.O.B. wrote the manuscript with help from all authors.

Competing interests

M.H.P. is a consultant and has equity interest in CRISPR Tx, but CRISPR Tx had no input into the design, execution, interpretation or publication of the results herein. N.U. was a former employee of Stem Cells, Inc., but they had no input into this manuscript.

Corresponding author

Correspondence to Matthew H. Porteus.

Reviewer Information Nature thanks B. Ebert, L. Naldini and the other anonymous reviewer(s) for their contribution to the peer review of this work.

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https://doi.org/10.1038/nature20134

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