CRISPR/Cas9 β-globin gene targeting in human haematopoietic stem cells

Daniel P. Dever, Rasmus O. Bak, Andreas Reinisch, Joab Camarena, Gabriel Washington, Carmencita E. Nicolas, Mara Pavel-Dinu, Nivi Saxena, Alec B. Wilkens, Sruthi Mantri, Nobuko Uchida, Ayal Hendel, Anupama Narla, Ravindra Majeti, Kenneth I. Weinberg and Matthew H. Porteus ()
Additional contact information
Daniel P. Dever: Stanford University
Rasmus O. Bak: Stanford University
Andreas Reinisch: Cancer Institute, and Institute for Stem Cell Biology and Regenerative Medicine, Stanford University
Joab Camarena: Stanford University
Gabriel Washington: Stanford University
Carmencita E. Nicolas: Stanford University
Mara Pavel-Dinu: Stanford University
Nivi Saxena: Stanford University
Alec B. Wilkens: Stanford University
Sruthi Mantri: Stanford University
Nobuko Uchida: Stem Cells, Inc. 7707 Gateway Blvd., Suite 140
Ayal Hendel: Stanford University
Anupama Narla: Stanford University School of Medicine
Ravindra Majeti: Cancer Institute, and Institute for Stem Cell Biology and Regenerative Medicine, Stanford University
Kenneth I. Weinberg: Stanford University
Matthew H. Porteus: Stanford University

Nature, 2016, vol. 539, issue 7629, 384-389

Abstract: 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.

Date: 2016
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DOI: 10.1038/nature20134

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