High-level correction of the sickle mutation is amplified in vivo during erythroid differentiation.

Publication Year: 
Wendy Magis
Mark A DeWitt
Stacia K Wyman
Jonathan T Vu
Seok-Jin Heo
Shirley J Shao
Finn Hennig
Zulema G Romero
Beatriz Campo-Fernandez
Suzanne Said
Matthew S McNeill
Garrett R Rettig
Yongming Sun
Yu Wang
Mark A Behlke
Donald B Kohn
Dario Boffelli
Mark C Walters
Jacob E Corn
David I K Martin
PubMed link: 
Public Summary: 
An optimized manufacturing protocol was developed and summarized in this report. The CRISPR gene editing tools generated a repaired sickle mutation in an average of 30% of stem cells from patients with sickle cell disease. This is a level that should be enough to have a clinical benefit and can be tested in a clinical trial.
Scientific Abstract: 
Background: A point mutation in sickle cell disease (SCD) alters one amino acid in the beta-globin subunit of hemoglobin, with resultant anemia and multiorgan damage that typically shortens lifespan by decades. Because SCD is caused by a single mutation, and hematopoietic stem cells (HSCs) can be harvested, manipulated, and returned to an individual, it is an attractive target for gene correction. Results: An optimized Cas9 ribonucleoprotein (RNP) with an ssDNA oligonucleotide donor together generated correction of at least one beta-globin allele in more than 30% of long-term engrafting human HSCs. After adopting a high-fidelity Cas9 variant, efficient correction with minimal off-target events also was observed. In vivo erythroid differentiation markedly enriches for corrected beta-globin alleles, indicating that erythroblasts carrying one or more corrected alleles have a survival advantage. Significance: These findings indicate that the sickle mutation can be corrected in autologous HSCs with an optimized protocol suitable for clinical translation.