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.

中文翻译：

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造血干细胞的高保真 PAMless 碱基编辑治疗慢性肉芽肿病


X 连锁慢性肉芽肿病 （X-CGD） 是由细胞色素 b-245 β 链 （CYBB） 基因基因突变引起的先天性免疫缺陷 （IEI）。碱基编辑器 （BE） 对纠正导致 X-CGD 的突变的适用性受到 Cas 酶识别特定原间隔区相邻基序 （PAM） 的要求的限制。我们最近设计的 PAMless Cas 酶 SpRY 可以克服 PAM 的限制。然而，基于 SpRY 的 BE 在纠正人类造血干细胞和祖细胞 （HSPC） 突变方面的效率、特异性和适用性尚未得到彻底检查。在这里，我们证明腺嘌呤 BE ABE8e-SpRY 可以访问 HSPC 中的一系列靶位点，以纠正导致 X-CGD 的突变。对于原型 X-CGD 突变 CYBB c.676C>T，ABE8e-SpRY 实现了高达 70% 的校正，效率比以前的 CRISPR 核酸酶和供体模板方法高出三倍半以上。我们分析了碱基编辑的 HSPC 中潜在的脱靶 DNA 编辑、转录组范围的 RNA 编辑和染色体扰动，这些共同揭示了最小的脱靶或旁观者编辑。将碱基编辑的 HSPCs 移植到免疫缺陷小鼠体内后，编辑的等位基因仍然存在。总之，这些研究性新药研究表明，使用 PAMless BE 可以有效、精确地纠正 X-CGD 突变，支持首次人体临床试验 （NCT06325709），并为治疗其他 IEI 突变提供潜在蓝图。