Luthers Christopher R, Mittelhauser Annika, Colamartino Aurelien, Wu Xiaomeng, Cirigliano Samuel, Long Joseph D, Sanchez Julie M, Romero Zulema, Kohn Donald B
Molecular Biology Interdepartmental Program, University of California, Los Angeles, Los Angeles, CA 90095, USA.
Department of Microbiology, Immunology and Molecular Genetics, University of California, Los Angeles, Los Angeles, CA 90095, USA.
Mol Ther Methods Clin Dev. 2025 Aug 12;33(3):101555. doi: 10.1016/j.omtm.2025.101555. eCollection 2025 Sep 11.
X-linked agammaglobulinemia (XLA) is a rare inborn error of immunity caused by loss-of-function mutations in the gene encoding Bruton's tyrosine kinase (BTK). XLA patients lack mature B cells and have negligible antibody levels, leaving them susceptible to recurrent bacterial and chronic viral infections. Autologous hematopoietic stem cell gene therapy with gene-corrected HSC may serve as a promising treatment of XLA; this therapy would provide a one-time cure and would replace lifelong immunoglobulin replacement therapy. Due to the requirement of strict physiological regulation of gene expression, a site-specific editing strategy was designed to insert a cDNA transgene directly into its endogenous locus. To study the effectiveness of this therapy, murine lineage-negative hematopoietic cells from a murine model of XLA were edited using CRISPR-Cas9/rAAV6 then transplanted into recipient XLA mice. Myeloablated XLA mice that received transplantation of Btk-corrected Lin- cells displayed high levels of engraftment, significant increases in their B cell levels, increased production of various immunoglobulins, improved B cell development in the bone marrow, increased B cell receptor diversity, and the ability to produce antigen-specific antibodies following immunization. Collectively, we have modeled a gene therapy strategy in a disease model of XLA and extensively validated the site-specific genome editing approach.
X连锁无丙种球蛋白血症（XLA）是一种罕见的先天性免疫缺陷病，由编码布鲁顿酪氨酸激酶（BTK）的基因功能丧失性突变引起。XLA患者缺乏成熟B细胞，抗体水平可忽略不计，因此易患复发性细菌感染和慢性病毒感染。用基因校正的造血干细胞进行自体造血干细胞基因治疗可能是一种有前景的XLA治疗方法；这种治疗将提供一次性治愈，并取代终身免疫球蛋白替代疗法。由于基因表达需要严格的生理调控，因此设计了一种位点特异性编辑策略，将cDNA转基因直接插入其内源性位点。为了研究这种治疗方法的有效性，使用CRISPR-Cas9/rAAV6对XLA小鼠模型的鼠系阴性造血细胞进行编辑，然后移植到受体XLA小鼠体内。接受Btk校正的Lin-细胞移植的经清髓处理的XLA小鼠显示出高嵌合率、B细胞水平显著增加、各种免疫球蛋白产量增加、骨髓中B细胞发育改善、B细胞受体多样性增加以及免疫后产生抗原特异性抗体的能力。总体而言，我们在XLA疾病模型中模拟了一种基因治疗策略，并广泛验证了位点特异性基因组编辑方法。
