Department of Pediatrics, Stanford University School of Medicine, Stanford, CA, USA.
Gene Therapy Center, Hospital de Clinicas de Porto Alegre, Porto Alegre, Brazil.
Nat Commun. 2020 Jul 3;11(1):3327. doi: 10.1038/s41467-020-17148-x.
Gaucher disease is a lysosomal storage disorder caused by insufficient glucocerebrosidase activity. Its hallmark manifestations are attributed to infiltration and inflammation by macrophages. Current therapies for Gaucher disease include life-long intravenous administration of recombinant glucocerebrosidase 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 glucocerebrosidase-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.
戈谢病是一种溶酶体贮积症，由葡萄糖脑苷脂酶活性不足引起。其典型表现归因于巨噬细胞的浸润和炎症。戈谢病的当前治疗方法包括终生静脉内给予重组葡萄糖脑苷脂酶和口服葡萄糖神经酰胺合酶抑制剂。另一种方法是对患者自身的造血系统进行工程改造，以恢复葡萄糖脑苷脂酶的表达，从而取代受影响的细胞，并构成该病的潜在一次性治疗方法。在这里，我们报告了一种高效的基于 CRISPR/Cas9 的方法，该方法使用单核细胞/巨噬细胞特异性元件将葡萄糖脑苷脂酶表达盒靶向到人类造血干/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酶的巨噬细胞，并通过连续移植保持长期的再群体化和多谱系分化潜能。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，并规避了干细胞中外源葡萄糖脑苷脂酶的潜在毒性。此外，它还构成了其他溶酶体酶缺乏症的适应性平台。
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