Characterization of Gene Alterations following Editing of the β-Globin Gene Locus in Hematopoietic Stem/Progenitor Cells

Joseph Long; Megan D Hoban; Aaron R Cooper; Michael L Kaufman; Caroline Y Kuo; Beatriz Campo-Fernandez; Dianne Lumaquin; Roger P Hollis; Xiaoyan Wang; Donald B Kohn; Zulema Romero

doi:10.1016/j.ymthe.2017.11.001

Characterization of Gene Alterations following Editing of the β-Globin Gene Locus in Hematopoietic Stem/Progenitor Cells

Mol Ther. 2018 Feb 7;26(2):468-479. doi: 10.1016/j.ymthe.2017.11.001. Epub 2017 Nov 9.

Authors

Affiliations

¹ Department of Microbiology, Immunology and Molecular Genetics, University of California, Los Angeles, Los Angeles, CA 90095, USA; Biology Department, California State University, Northridge, Northridge, CA 91330, USA.
² Department of Microbiology, Immunology and Molecular Genetics, University of California, Los Angeles, Los Angeles, CA 90095, USA.
³ Molecular Biology Interdepartmental Ph.D. Program, University of California, Los Angeles, Los Angeles, CA 90095, USA.
⁴ Division of Allergy and Immunology, Department of Pediatrics, David Geffen School of Medicine, University of California, Los Angeles, Los Angeles, CA 90095, USA.
⁵ Department of Internal Medicine and Health Services Research, University of California, Los Angeles, Los Angeles, CA 90095, USA.
⁶ Department of Microbiology, Immunology and Molecular Genetics, University of California, Los Angeles, Los Angeles, CA 90095, USA; Eli & Edythe Broad Center of Regenerative Medicine & Stem Cell Research, University of California, Los Angeles, Los Angeles, CA 90095, USA.
⁷ Department of Microbiology, Immunology and Molecular Genetics, University of California, Los Angeles, Los Angeles, CA 90095, USA. Electronic address: zulemar@ucla.edu.

Abstract

The use of engineered nucleases combined with a homologous DNA donor template can result in targeted gene correction of the sickle cell disease mutation in hematopoietic stem and progenitor cells. However, because of the high homology between the adjacent human β- and δ-globin genes, off-target cleavage is observed at δ-globin when using some endonucleases targeted to the sickle mutation in β-globin. Introduction of multiple double-stranded breaks by endonucleases has the potential to induce intergenic alterations. Using a novel droplet digital PCR assay and high-throughput sequencing, we characterized the frequency of rearrangements between the β- and δ-globin paralogs when delivering these nucleases. Pooled CD34⁺ cells and colony-forming units from sickle bone marrow were treated with nuclease only or including a donor template and then analyzed for potential gene rearrangements. It was observed that, in pooled CD34⁺ cells and colony-forming units, the intergenic β-δ-globin deletion was the most frequent rearrangement, followed by inversion of the intergenic fragment, with the inter-chromosomal translocation as the least frequent. No rearrangements were observed when endonuclease activity was restricted to on-target β-globin cleavage. These findings demonstrate the need to develop site-specific endonucleases with high specificity to avoid unwanted gene alterations.

Keywords: chromosomal rearrangement; endonucleases; off-target.

Publication types

Research Support, N.I.H., Extramural
Research Support, Non-U.S. Gov't
Research Support, U.S. Gov't, Non-P.H.S.

MeSH terms

Gene Conversion
Gene Editing*
Gene Rearrangement
Gene Targeting
Genetic Variation*
Hematopoietic Stem Cells / metabolism*
High-Throughput Nucleotide Sequencing
Humans
Nucleic Acid Amplification Techniques
Translocation, Genetic
beta-Globins / genetics*

Substances

beta-Globins

Abstract

Publication types

MeSH terms

Substances

Grants and funding