{"title":"Impact of CRISPR/HDR editing versus lentiviral transduction on long-term engraftment and clonal dynamics of HSPCs in rhesus macaques.","authors":"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","DOIUrl":null,"url":null,"abstract":"<p><p>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.</p>","PeriodicalId":93928,"journal":{"name":"Cell stem cell","volume":" ","pages":"455-466.e4"},"PeriodicalIF":0.0000,"publicationDate":"2024-04-04","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10997443/pdf/","citationCount":"0","resultStr":null,"platform":"Semanticscholar","paperid":null,"PeriodicalName":"Cell stem cell","FirstCategoryId":"1085","ListUrlMain":"https://doi.org/10.1016/j.stem.2024.02.010","RegionNum":0,"RegionCategory":null,"ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":null,"EPubDate":"2024/3/19 0:00:00","PubModel":"Epub","JCR":"","JCRName":"","Score":null,"Total":0}
引用次数: 0
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.