Vera Bzhilyanskaya, Linyuan Ma, Siyuan Liu, Lauren R. Fox, Madelynn N. Whittaker, Ronald J. Meis, Uimook Choi, Amanda Lawson, Michelle Ma, Narda Theobald, Sandra Burkett, Colin L. Sweeney, Cicera R. Lazzarotto, Shengdar Q. Tsai, Justin B. Lack, Xiaolin Wu, Gary A. Dahl, Harry L. Malech, Benjamin P. Kleinstiver, Suk See De Ravin
{"title":"High-fidelity PAMless base editing of hematopoietic stem cells to treat chronic granulomatous disease","authors":"Vera Bzhilyanskaya, Linyuan Ma, Siyuan Liu, Lauren R. Fox, Madelynn N. Whittaker, Ronald J. Meis, Uimook Choi, Amanda Lawson, Michelle Ma, Narda Theobald, Sandra Burkett, Colin L. Sweeney, Cicera R. Lazzarotto, Shengdar Q. Tsai, Justin B. Lack, Xiaolin Wu, Gary A. Dahl, Harry L. Malech, Benjamin P. Kleinstiver, Suk See De Ravin","doi":"10.1126/scitranslmed.adj6779","DOIUrl":null,"url":null,"abstract":"<div >X-linked chronic granulomatous disease (X-CGD) is an inborn error of immunity (IEI) resulting from genetic mutations in the cytochrome b-245 beta chain (<i>CYBB</i>) gene. The applicability of base editors (BEs) to correct mutations that cause X-CGD is constrained by the requirement of Cas enzymes to recognize specific protospacer adjacent motifs (PAMs). Our recently engineered PAMless Cas enzyme, SpRY, can overcome the PAM limitation. However, the efficiency, specificity, and applicability of SpRY-based BEs to correct mutations in human hematopoietic stem and progenitor cells (HSPCs) have not been thoroughly examined. Here, we demonstrated that the adenine BE ABE8e-SpRY can access a range of target sites in HSPCs to correct mutations causative of X-CGD. For the prototypical X-CGD mutation <i>CYBB</i> c.676C>T, ABE8e-SpRY achieved up to 70% correction, reaching efficiencies greater than three-and-one-half times higher than previous CRISPR nuclease and donor template approaches. We profiled potential off-target DNA edits, transcriptome-wide RNA edits, and chromosomal perturbations in base-edited HSPCs, which together revealed minimal off-target or bystander edits. Edited alleles persisted after transplantation of the base-edited HSPCs into immunodeficient mice. Together, these investigational new drug–enabling studies demonstrated efficient and precise correction of an X-CGD mutation with PAMless BEs, supporting a first-in-human clinical trial (NCT06325709) and providing a potential blueprint for treatment of other IEI mutations.</div>","PeriodicalId":21580,"journal":{"name":"Science Translational Medicine","volume":"16 769","pages":""},"PeriodicalIF":15.8000,"publicationDate":"2024-10-16","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":"0","resultStr":null,"platform":"Semanticscholar","paperid":null,"PeriodicalName":"Science Translational Medicine","FirstCategoryId":"3","ListUrlMain":"https://www.science.org/doi/10.1126/scitranslmed.adj6779","RegionNum":1,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":null,"EPubDate":"","PubModel":"","JCR":"Q1","JCRName":"CELL BIOLOGY","Score":null,"Total":0}
引用次数: 0
Abstract
X-linked chronic granulomatous disease (X-CGD) is an inborn error of immunity (IEI) resulting from genetic mutations in the cytochrome b-245 beta chain (CYBB) gene. The applicability of base editors (BEs) to correct mutations that cause X-CGD is constrained by the requirement of Cas enzymes to recognize specific protospacer adjacent motifs (PAMs). Our recently engineered PAMless Cas enzyme, SpRY, can overcome the PAM limitation. However, the efficiency, specificity, and applicability of SpRY-based BEs to correct mutations in human hematopoietic stem and progenitor cells (HSPCs) have not been thoroughly examined. Here, we demonstrated that the adenine BE ABE8e-SpRY can access a range of target sites in HSPCs to correct mutations causative of X-CGD. For the prototypical X-CGD mutation CYBB c.676C>T, ABE8e-SpRY achieved up to 70% correction, reaching efficiencies greater than three-and-one-half times higher than previous CRISPR nuclease and donor template approaches. We profiled potential off-target DNA edits, transcriptome-wide RNA edits, and chromosomal perturbations in base-edited HSPCs, which together revealed minimal off-target or bystander edits. Edited alleles persisted after transplantation of the base-edited HSPCs into immunodeficient mice. Together, these investigational new drug–enabling studies demonstrated efficient and precise correction of an X-CGD mutation with PAMless BEs, supporting a first-in-human clinical trial (NCT06325709) and providing a potential blueprint for treatment of other IEI mutations.
期刊介绍:
Science Translational Medicine is an online journal that focuses on publishing research at the intersection of science, engineering, and medicine. The goal of the journal is to promote human health by providing a platform for researchers from various disciplines to communicate their latest advancements in biomedical, translational, and clinical research.
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