Enhanced homology-directed repair for highly efficient gene editing in hematopoietic stem/progenitor cells

• Published in: Blood Vol. 137; no. 19; pp. 2598 - 2608
• Main Authors: De Ravin, Suk See, Brault, Julie, Meis, Ronald J., Liu, Siyuan, Li, Linhong, Pavel-Dinu, Mara, Lazzarotto, Cicera R., Liu, Taylor, Koontz, Sherry M., Choi, Uimook, Sweeney, Colin L., Theobald, Narda, Lee, GaHyun, Clark, Aaron B., Burkett, Sandra S., Kleinstiver, Benjamin P., Porteus, Matthew H., Tsai, Shengdar, Kuhns, Douglas B., Dahl, Gary A., Headey, Stephen, Wu, Xiaolin, Malech, Harry L.
• Format: Journal Article
• Language: English
• Published: United States Elsevier Inc 13.05.2021; American Society of Hematology
• Subjects: Animals; Bacterial Proteins; Caspase 9; Cells, Cultured; Dependovirus - genetics; DNA Repair - genetics; Exons - genetics; Gene Editing - methods; Gene Therapy; Genetic Therapy - methods; Genetic Vectors - genetics; Genetic Vectors - therapeutic use; Granulomatous Disease, Chronic - genetics; Granulomatous Disease, Chronic - therapy; Hematopoietic Stem Cell Transplantation; Hematopoietic Stem Cells - enzymology; Heterografts; Humans; Male; Mice; Mice, Inbred NOD; Mice, SCID; NADPH Oxidase 2 - deficiency; NADPH Oxidase 2 - genetics; Phagocytes - metabolism; Reactive Oxygen Species; Ribonucleoproteins - genetics; RNA, Messenger - genetics; Sequence Deletion; Streptococcus pyogenes - enzymology; Tumor Suppressor p53-Binding Protein 1 - antagonists & inhibitors
• ISSN: 0006-4971; 1528-0020; 1528-0020
• DOI: 10.1182/blood.2020008503

Lentivector gene therapy for X-linked chronic granulomatous disease (X-CGD) has proven to be a viable approach, but random vector integration and subnormal protein production from exogenous promoters in transduced cells remain concerning for long-term safety and efficacy. A previous genome editing–based approach using Streptococcus pyogenes Cas9 mRNA and an oligodeoxynucleotide donor to repair genetic mutations showed the capability to restore physiological protein expression but lacked sufficient efficiency in quiescent CD34+ hematopoietic cells for clinical translation. Here, we report that transient inhibition of p53-binding protein 1 (53BP1) significantly increased (2.3-fold) long-term homology-directed repair to achieve highly efficient (80% gp91phox+ cells compared with healthy donor control subjects) long-term correction of X-CGD CD34+ cells. •CRISPR/Cas9 repair of CYBB mutations with an oligodeoxynucleotide restores endogenous regulation of expression in X-CGD CD34+ cells.•Gene-editing enhancers greatly increase homology-directed repair to achieve highly efficient gene correction of engrafting CD34+ cells. [Display omitted]