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

Publication Type	Academic Article
Authors	Long J, Hoban M, Cooper A, Kaufman M, Kuo C, Campo-Fernandez B, Lumaquin D, Hollis R, Wang X, Kohn D, Romero Z
Journal	Mol Ther
Volume	26
Issue	2
Pagination	468-479
Date Published	11/09/2017
ISSN	1525-0024
Keywords	Gene Editing, Genetic Variation, Hematopoietic Stem Cells, beta-Globins
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.
DOI	10.1016/j.ymthe.2017.11.001
PubMed ID	29221806
PubMed Central ID	PMC5835144