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Correction of X-CGD patient HSPCs by targeted CYBB cDNA insertion using CRISPR/Cas9 with 53BP1 inhibition for enhanced homology-directed repair

Gene Therapy volume 28, pages 373–390 (2021)Cite this article

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Abstract

X-linked chronic granulomatous disease is an immunodeficiency characterized by defective production of microbicidal reactive oxygen species (ROS) by phagocytes. Causative mutations occur throughout the 13 exons and splice sites of the CYBB gene, resulting in loss of gp91phox protein. Here we report gene correction by homology-directed repair in patient hematopoietic stem/progenitor cells (HSPCs) using CRISPR/Cas9 for targeted insertion of CYBB exon 1–13 or 2–13 cDNAs from adeno-associated virus donors at endogenous CYBB exon 1 or exon 2 sites. Targeted insertion of exon 1–13 cDNA did not restore physiologic gp91phox levels, consistent with a requirement for intron 1 in CYBB expression. However, insertion of exon 2–13 cDNA fully restored gp91phox and ROS production upon phagocyte differentiation. Addition of a woodchuck hepatitis virus post-transcriptional regulatory element did not further enhance gp91phox expression in exon 2–13 corrected cells, indicating that retention of intron 1 was sufficient for optimal CYBB expression. Targeted correction was increased ~1.5-fold using i53 mRNA to transiently inhibit nonhomologous end joining. Following engraftment in NSG mice, corrected HSPCs generated phagocytes with restored gp91phox and ROS production. Our findings demonstrate the utility of tailoring donor design and targeting strategies to retain regulatory elements needed for optimal expression of the target gene.

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Fig. 1: Schemata of correction strategies for targeted CYBB cDNA insertion in X-CGD patient CD34+ HSPCs.
Fig. 2: Targeted genome editing efficiencies in HSPCs for correction strategies 1 through 3 using initial un-optimized editing conditions.
Fig. 3: Comparison of phenotypic correction of gp91phox expression in differentiated phagocytes after targeted cDNA insertion in X-CGD HSPCs.
Fig. 4: Comparison of functional correction of ROS production measured by DHR assay in differentiated phagocytes after targeted cDNA insertion in X-CGD HSPCs.
Fig. 5: Enhancement of targeted insertion efficiency using i53 mRNA in HSPCs with correction strategy 3.
Fig. 6: Engraftment data of X-CGD patient HSPCs corrected using strategy 2 with optimized targeted insertion conditions and transplanted into NSG mice.

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Acknowledgements

We thank the patients and healthy donors for their contribution to this study, the Department of Transfusion Medicine at the National Institutes of Health Clinical Center for their collection and processing of CD34+ HSPCs, and the CCR Genomics Core of the National Cancer Institute for their assistance in DNA sequencing and ddPCR analyses.

Funding

This research was supported by the Intramural Research Program of the National Institute of Allergy and Infectious Diseases, National Institutes of Health (NIH) under intramural project numbers Z01-Al-00644 and Z01-Al-00988, as well as funding to MHP under NIH research grant R01-AI097320 and philanthropic gift funds from the Amon G. Carter Foundation.

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  1. These authors contributed equally: Colin L. Sweeney, Mara Pavel-Dinu

Authors and Affiliations

  1. Genetic Immunotherapy Section, Laboratory of Clinical Immunology and Microbiology, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD, USA

    Colin L. Sweeney, Uimook Choi, Julie Brault, Taylor Liu, Sherry Koontz, Narda Theobald, Janet Lee, Ezekiel A. Bello, Harry L. Malech & Suk See De Ravin

  2. Department of Pediatrics, Stanford University School of Medicine, Stanford, CA, USA

    Mara Pavel-Dinu & Matthew H. Porteus

  3. MaxCyte Inc., Gaithersburg, MD, USA

    Linhong Li

  4. Cancer Research Technology Program, Leidos Biomedical Research Inc., Frederick, MD, USA

    Xiaolin Wu

  5. CELLSCRIPT, LLC, Madison, WI, USA

    Ronald J. Meis & Gary A. Dahl

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LL was a full-time employee of MaxCyte Biosystems at the time of this study; RJM and GAD are full-time employees of CELLSCRIPT, LLC; MHP holds equity in CRISPR Tx and Allogene Tx and serves on the Scientific Advisory Board for Allogene Tx; the remaining authors declare no competing interests.

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Sweeney, C.L., Pavel-Dinu, M., Choi, U. et al. Correction of X-CGD patient HSPCs by targeted CYBB cDNA insertion using CRISPR/Cas9 with 53BP1 inhibition for enhanced homology-directed repair. Gene Ther 28, 373–390 (2021). https://doi.org/10.1038/s41434-021-00251-z

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