Gene therapy in hematopoietic stem and progenitor cells (HSPCs) shows great potential for the treatment of inborn metabolic diseases. Typical HSPC gene therapy approaches rely on constitutive promoters to express a therapeutic transgene, which is associated with multiple disadvantages. Here, we propose a novel promoterless intronic gene editing approach that triggers transgene expression only after cellular differentiation into the myeloid lineage. We integrated a splicing-competent eGFP cassette into the first intron of and observed expression of eGFP in the myeloid lineage but minimal to no expression in HSPCs or differentiated non-myeloid lineages. , edited HSPCs successfully engrafted in immunodeficient mice and displayed transgene expression in the myeloid compartment of multiple tissues. Using the same approach, we expressed alpha-L-iduronidase (IDUA), the defective enzyme in Mucopolysaccharidosis type I, and observed a 10-fold supraendogenous IDUA expression exclusively after myeloid differentiation. Edited cells efficiently populated bone marrow, blood, and spleen of immunodeficient mice, and retained the capacity to secrete IDUA . Importantly, cells edited with the eGFP and IDUA transgenes were also found in the brain. This approach may unlock new therapeutic strategies for inborn metabolic and neurological diseases that require the delivery of therapeutics in brain.

中文翻译：

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TALEN 介导的 HSPC 内含子编辑使转基因表达仅限于骨髓谱系


造血干细胞和祖细胞（HSPC）的基因治疗显示出治疗先天代谢疾病的巨大潜力。典型的 HSPC 基因治疗方法依赖于组成型启动子来表达治疗性转基因，这与多种缺点相关。在这里，我们提出了一种新的无启动子内含子基因编辑方法，仅在细胞分化为骨髓谱系后才触发转基因表达。我们将具有剪接能力的 eGFP 盒整合到第一个内含子中，并观察到 ​​eGFP 在骨髓谱系中表达，但在 HSPC 或分化的非骨髓谱系中表达很少或没有表达。 ，编辑后的 ​​HSPC 成功植入免疫缺陷小鼠体内，并在多个组织的骨髓区室中显示转基因表达。使用相同的方法，我们表达了 α-L-艾杜糖醛酸酶 (IDUA)，这是 I 型粘多糖贮积症中的缺陷酶，并观察到仅在骨髓分化后 10 倍的超内源性 IDUA 表达。编辑后的细胞有效地填充了免疫缺陷小鼠的骨髓、血液和脾脏，并保留了分泌 IDUA 的能力。重要的是，在大脑中也发现了用 eGFP 和 IDUA 转基因编辑的细胞。这种方法可能会为需要在大脑中提供治疗的先天性代谢和神经系统疾病解锁新的治疗策略。