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

• Published in: Science translational medicine 2024-10, Vol.16 (769), p.eadj6779
• Main Authors: Bzhilyanskaya, Vera, Ma, Linyuan, Liu, Siyuan, Fox, Lauren R, Whittaker, Madelynn N, Meis, Ronald J, Choi, Uimook, Lawson, Amanda, Ma, Michelle, Theobald, Narda, Burkett, Sandra, Sweeney, Colin L, Lazzarotto, Cicera R, Tsai, Shengdar Q, Lack, Justin B, Wu, Xiaolin, Dahl, Gary A, Malech, Harry L, Kleinstiver, Benjamin P, De Ravin, Suk See
• Format: Article
• Language: English
• Subjects: Animals; Chronic granulomatous disease; CRISPR; CRISPR-Cas Systems - genetics; Cytochrome b; Gene Editing - methods; Granulomatous Disease, Chronic - genetics; Granulomatous Disease, Chronic - therapy; Hematopoietic Stem Cell Transplantation; Hematopoietic stem cells; Hematopoietic Stem Cells - metabolism; Hemopoiesis; Humans; Immunodeficiency; Mice; Mutation; Mutation - genetics; NADPH Oxidase 2 - genetics; NADPH Oxidase 2 - metabolism; Progenitor cells; Transcriptomes
• ISSN: 1946-6234; 1946-6242; 1946-6242; 1946-3242
• DOI: 10.1126/scitranslmed.adj6779

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 ( ) 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 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.