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

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

doi:10.1016/j.isci.2022.104374

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

iScience. 2022 May 10;25(6):104374. doi: 10.1016/j.isci.2022.104374. eCollection 2022 Jun 17.

Authors

Wendy Magis¹, Mark A DeWitt², Stacia K Wyman², Jonathan T Vu², Seok-Jin Heo¹, Shirley J Shao², Finn Hennig³, Zulema G Romero⁴, Beatriz Campo-Fernandez⁴, Suzanne Said⁴, Matthew S McNeill⁵, Garrett R Rettig⁵, Yongming Sun⁶, Yu Wang⁶, Mark A Behlke⁵, Donald B Kohn⁴, Dario Boffelli¹, Mark C Walters^{1

7}, Jacob E Corn^{2

8

9}, David I K Martin¹

Affiliations

¹ Children's Hospital Oakland Research Institute, UCSF Benioff Children's Hospital Oakland, Oakland, CA 94609, USA.
² Innovative Genomics Institute, University of California, Berkeley, CA 94720, USA.
³ Jacob's School of Medicine and Biomedical Sciences, Buffalo, NY 14203, USA.
⁴ Departments of Microbiology, Immunology, and Molecular Genetics, University of California, Los Angeles, CA 90095, USA.
⁵ Integrated DNA Technologies, Coralville, IA 52241, USA.
⁶ Integrated DNA Technologies, Redwood City, CA 94065, USA.
⁷ Blood and Marrow Transplant Program, Division of Hematology, UCSF Benioff Children's Hospital Oakland, Oakland, CA 94609, USA.
⁸ Department of Molecular and Cell Biology, University of California, Berkeley, CA 94720, USA.
⁹ Department of Biology, ETH Zurich, Zurich 8093, Switzerland.

Abstract

Background: A point mutation in sickle cell disease (SCD) alters one amino acid in the β-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 β-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 β-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.

Keywords: genetic engineering; genetics; molecular genetics.