Gaucher disease is a lysosomal storage disorder caused by insufficient glucocerebroside activity. Its hallmark manifestations are attributed to infiltration and inflammation by macrophages. Current therapies for Gaucher disease include life−long intravenous administration of recombinant glucocerebroside and orally-available glucosylceramide synthase inhibitors. An alternative approach is to engineer the patient’s own hematopoietic system to restore glucocerebrosidase expression, thereby replacing the affected cells, and constituting a potential one-time therapy for this disease. Here, we report an efficient CRISPR/Cas9-based approach that targets glucocerebrosidase expression cassettes with a monocyte/macrophage-specific element to the CCR5 safe-harbor locus in human hematopoietic stem and progenitor cells. The targeted cells generate glucocerebroside-expressing macrophages and maintain long-term repopulation and multi-lineage differentiation potential with serial transplantation. The combination of a safe-harbor and a lineage-specific promoter establishes a universal correction strategy and circumvents potential toxicity of ectopic glucocerebrosidase in the stem cells. Furthermore, it constitutes an adaptable platform for other lysosomal enzyme deficiencies.

戈谢病是一种由葡萄糖脑苷脂活性不足引起的溶酶体贮积症。其标志性表现归因于巨噬细胞的浸润和炎症。目前治疗戈谢病的方法包括终生静脉注射重组葡萄糖脑苷脂和口服葡萄糖神经酰胺合酶抑制剂。另一种方法是改造患者自身的造血系统以恢复葡萄糖脑苷脂酶的表达，从而替换受影响的细胞，并构成对该疾病的潜在一次性疗法。在这里，我们报告了一种有效的基于 CRISPR/Cas9 的方法，该方法将具有单核细胞/巨噬细胞特异性元件的葡糖脑苷脂酶表达盒靶向人类造血干细胞和祖细胞中的 CCR5 安全港基因座。靶向细胞产生表达葡萄糖脑苷脂的巨噬细胞，并通过连续移植维持长期的再增殖和多向分化潜能。安全港和谱系特异性启动子的组合建立了一种通用的校正策略，并规避了异位葡萄糖脑苷脂酶在干细胞中的潜在毒性。此外，它构成了其他溶酶体酶缺陷的适应性平台。