Impact of CRISPR/HDR editing versus lentiviral transduction on long-term engraftment and clonal dynamics of HSPCs in rhesus macaques

Byung-Chul Lee; Ashley Gin; Chuanfeng Wu; Komudi Singh; Max Grice; Ryland Mortlock; Diana Abraham; Xing Fan; Yifan Zhou; Aisha AlJanahi; Uimook Choi; Suk See DeRavin; Taehoon Shin; Sogun Hong; Cynthia E Dunbar

doi:10.1016/j.stem.2024.02.010

Impact of CRISPR/HDR editing versus lentiviral transduction on long-term engraftment and clonal dynamics of HSPCs in rhesus macaques

Cell Stem Cell. 2024 Apr 4;31(4):455-466.e4. doi: 10.1016/j.stem.2024.02.010. Epub 2024 Mar 19.

Authors

Affiliations

¹ Translational Stem Cell Biology Branch, National Heart Lung and Blood Institute, National Institutes of Health, Bethesda, MD, USA; Department of Biological Sciences, Sookmyung Women's University, Seoul, Korea; Research Institute of Women's Health, Sookmyung Women's University, Seoul, Korea. Electronic address: bclee@sookmyung.ac.kr.
² Translational Stem Cell Biology Branch, National Heart Lung and Blood Institute, National Institutes of Health, Bethesda, MD, USA.
³ Translational Stem Cell Biology Branch, National Heart Lung and Blood Institute, National Institutes of Health, Bethesda, MD, USA; Wellcome-MRC Cambridge Stem Cell Institute, University of Cambridge, Puddicombe Way, Cambridge, UK; Wellcome Sanger Institute, Wellcome Genome Campus, Hinxton, Cambridge CB10 1SA, UK.
⁴ Laboratory of Clinical Immunology and Microbiology, National Institute of Allergy and Infectious Diseases, NIH, Bethesda, MD, USA.
⁵ Translational Stem Cell Biology Branch, National Heart Lung and Blood Institute, National Institutes of Health, Bethesda, MD, USA; Department of Laboratory Animal Medicine, College of Veterinary Medicine, Jeju National University, Jeju, Korea.
⁶ Translational Stem Cell Biology Branch, National Heart Lung and Blood Institute, National Institutes of Health, Bethesda, MD, USA. Electronic address: dunbarc@nhlbi.nih.gov.

PMID: 38508195
PMCID: PMC10997443 (available on 2025-04-04)
DOI: 10.1016/j.stem.2024.02.010

Abstract

For precise genome editing via CRISPR/homology-directed repair (HDR), effective and safe editing of long-term engrafting hematopoietic stem cells (LT-HSCs) is required. The impact of HDR on true LT-HSC clonal dynamics in a relevant large animal model has not been studied. To track the output and clonality of HDR-edited cells and to provide a comparison to lentivirally transduced HSCs in vivo, we developed a competitive rhesus macaque (RM) autologous transplantation model, co-infusing HSCs transduced with a barcoded GFP-expressing lentiviral vector (LV) and HDR edited at the CD33 locus. CRISPR/HDR-edited cells showed a two-log decrease by 2 months following transplantation, with little improvement via p53 inhibition, in comparison to minimal loss of LV-transduced cells long term. HDR long-term clonality was oligoclonal in contrast to highly polyclonal LV-transduced HSCs. These results suggest marked clinically relevant differences in the impact of current genetic modification approaches on HSCs.

Keywords: CRISPR-Cas editing; HSC gene therapy; ex vivo; genetic barcoding; hematopoietic stem cells; homology-directed repair; lentiviral gene addition; precise genome editing; rhesus macaque.

MeSH terms

Animals
CRISPR-Cas Systems / genetics
Clustered Regularly Interspaced Short Palindromic Repeats
Gene Editing / methods
Hematopoietic Stem Cell Transplantation* / methods
Hematopoietic Stem Cells
Lentivirus / genetics
Macaca mulatta / genetics

Abstract

MeSH terms

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