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.
| Original language | English |
|---|---|
| Pages (from-to) | 384-389 |
| Number of pages | 6 |
| Journal | Nature |
| Volume | 539 |
| Issue number | 7629 |
| DOIs | |
| State | Published - 17 Nov 2016 |
| Externally published | Yes |
Bibliographical note
Publisher Copyright:© 2016 Macmillan Publishers Limited.
Funding
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.
| Funders | Funder number |
|---|---|
| Binns Program for Cord Blood Research | |
| Laurie Kraus Lacob Faculty Scholar Award in Pediatric Translational Research | |
| Medical Sciences | |
| National Institutes of Health | R01-AI097320, R01-AI120766 |
| National Eye Institute | PN2EY018244 |
| Amon G. Carter Foundation | |
| Stanford University | |
| Natur og Univers, Det Frie Forskningsråd | |
| Children's Health Research Institute | DFF-1331-00735B, DFF-1333-00106B |
| Stanford Maternal and Child Health Research Institute |