High-fidelity PAMless base editing of hematopoietic stem cells to treat chronic granulomatous disease

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.

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Publication Details

Journal
Science Translational Medicine
Published
2024-10-16
DOI
https://doi.org/10.1126/scitranslmed.adj6779
Citations
15
Primary Topic
CRISPR and Genetic Engineering
Type
article
Field-Weighted Citation Impact
3.51

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article

High-fidelity PAMless base editing of hematopoietic stem cells to treat chronic granulomatous disease

Madelynn N. Whittaker, Xiaolin Wu, Harry L. Malech, Narda Theobald et al.
15 citations
Science Translational Medicine
CRISPR and Genetic Engineering
3.51
article

High-fidelity PAMless base editing of hematopoietic stem cells to treat chronic granulomatous disease

Madelynn N. Whittaker, Xiaolin Wu, Harry L. Malech, Narda Theobald, Michelle Ma, Cícera R. Lazzarotto, Justin Lack, Colin L. Sweeney, Vera Bzhilyanskaya, Uimook Choi, Gary A. Dahl, Benjamin P. Kleinstiver, Lauren R. Fox, Sandra Burkett, Linyuan Ma, Suk See De Ravin, Shengdar Q. Tsai, Siyuan Liu, A. A. H. Lawson, Ronald J. Meis
article en
15 citations

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 (<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.

Science Translational MedicineVol. 16(769)
Leidos (United States) (US), St. Jude Children's Research Hospital (US), Harvard University (US), Wisconsin Institutes for Discovery (US), Massachusetts General Hospital (US), Allscripts (United States) (US), Frederick National Laboratory for Cancer Research (US), National Institute of Allergy and Infectious Diseases (US), National Cancer Institute (US)
Massachusetts General Hospital, St. Jude Children's Research Hospital, National Institutes of Health, National Cancer Institute, National Institute of Allergy and Infectious Diseases
Good health and well-being
Openalex Percentile: Top 6%
CRISPR and Genetic Engineering
3.51
DOIOpen Access PDFOpenAlex
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