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

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.

Automated summary

This study demonstrates the potential for gene correction of sickle cell disease by optimizing the use of Cas9 ribonucleoprotein and ssDNA oligonucleotide donor in autologous hematopoietic stem cells. The findings suggest a promising approach for clinical translation.