Infection, Immunity and Inflammation Research and Teaching Department, University College London Great Ormond Street Institute of Child Health, London, United Kingdom.
University College London Institute of Immunity and Transplantation, London, United Kingdom; Department of Immunology, Royal Free London National Health Service Foundation Trust, London, United Kingdom.
J Allergy Clin Immunol. 2024 Jul;154(1):195-208.e8. doi: 10.1016/j.jaci.2024.03.003. Epub 2024 Mar 11.
X-linked agammaglobulinemia (XLA) is an inborn error of immunity that renders boys susceptible to life-threatening infections due to loss of mature B cells and circulating immunoglobulins. It is caused by defects in the gene encoding the Bruton tyrosine kinase (BTK) that mediates the maturation of B cells in the bone marrow and their activation in the periphery. This paper reports on a gene editing protocol to achieve "knock-in" of a therapeutic BTK cassette in hematopoietic stem and progenitor cells (HSPCs) as a treatment for XLA.
To rescue BTK expression, this study employed a clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 9 system that creates a DNA double-strand break in an early exon of the BTK locus and an adeno-associated virus 6 virus that carries the donor template for homology-directed repair. The investigators evaluated the efficacy of the gene editing approach in HSPCs from patients with XLA that were cultured in vitro under B-cell differentiation conditions or that were transplanted in immunodeficient mice to study B-cell output in vivo.
A (feeder-free) B-cell differentiation protocol was successfully applied to blood-mobilized HSPCs to reproduce in vitro the defects in B-cell maturation observed in patients with XLA. Using this system, the investigators could show the rescue of B-cell maturation by gene editing. Transplantation of edited XLA HSPCs into immunodeficient mice led to restoration of the human B-cell lineage compartment in the bone marrow and immunoglobulin production in the periphery.
Gene editing efficiencies above 30% could be consistently achieved in human HSPCs. Given the potential selective advantage of corrected cells, as suggested by skewed X-linked inactivation in carrier females and by competitive repopulating experiments in mouse models, this work demonstrates the potential of this strategy as a future definitive therapy for XLA.
X 连锁无丙种球蛋白血症（XLA）是一种先天性免疫缺陷病，由于成熟 B 细胞和循环免疫球蛋白的缺失，男孩易发生危及生命的感染。它是由编码 Bruton 酪氨酸激酶（BTK）的基因缺陷引起的，BTK 介导骨髓中 B 细胞的成熟及其在外周的激活。本文报告了一种基因编辑方案，旨在将治疗性 BTK 盒“敲入”造血干细胞和祖细胞（HSPCs）中，作为 XLA 的治疗方法。
为了挽救 BTK 的表达，本研究采用了一种簇状规则间隔短回文重复序列/CRISPR 相关蛋白 9 系统，该系统在 BTK 基因座的早期外显子中产生 DNA 双链断裂，并使用携带同源定向修复供体模板的腺相关病毒 6 病毒。研究人员评估了该基因编辑方法在体外 B 细胞分化条件下培养的 XLA 患者 HSPCs 或移植到免疫缺陷小鼠中研究体内 B 细胞输出的疗效。
（无饲养层）B 细胞分化方案成功应用于血液动员的 HSPCs，以重现 XLA 患者观察到的 B 细胞成熟缺陷。使用该系统，研究人员可以证明基因编辑可以挽救 B 细胞成熟。编辑后的 XLA HSPCs 移植到免疫缺陷小鼠中，导致骨髓中人类 B 细胞谱系区室和外周免疫球蛋白的恢复。
在人类 HSPCs 中可以始终实现超过 30%的基因编辑效率。鉴于校正细胞的潜在选择性优势，如携带者女性中偏性 X 连锁失活和小鼠模型中的竞争性再定植实验所表明的那样，这项工作证明了该策略作为 XLA 未来确定性治疗方法的潜力。
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