Site-Specific Gene Editing of Human Hematopoietic Stem Cells for X-Linked Hyper-IgM Syndrome.
Site-Specific Gene Editing of Human Hematopoietic Stem Cells for X-Linked Hyper-IgM Syndrome.
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DOI：
10.1016/j.celrep.2018.04.103
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发表时间：
2018-05-29
期刊：
影响因子：
8.8
通讯作者：
Kohn DB
中科院分区：
文献类型：
--
作者：
Kuo CY;Long JD;Campo-Fernandez B;de Oliveira S;Cooper AR;Romero Z;Hoban MD;Joglekar AV;Lill GR;Kaufman ML;Fitz-Gibbon S;Wang X;Hollis RP;Kohn DB
X连锁高IgM综合征（XHIM）是一种原发性免疫缺陷，由于CD 40配体突变，影响免疫球蛋白类转换重组和体细胞高突变。这种疾病适合使用逆转录病毒载体进行基因治疗，但基因表达失调导致小鼠模型中的异常淋巴细胞增殖，突出了替代策略的必要性。在这里，我们证明了TALEN和CRISPR/Cas9平台有效驱动腺相关病毒递送的CD 40 L cDNA的正常拷贝整合的能力。供体序列在内源性CD 40 L启动子下游的位点特异性插入维持了CD 40 L的生理表达，同时覆盖了所有报告的下游突变。在原代人造血干细胞（HSC）以及细胞系和XHIM患者来源的T细胞中实现了高水平的基因修饰。值得注意的是，基因校正的HSC以临床相关的频率移植到免疫缺陷小鼠中。这些研究为XHIM的永久性治疗提供了基础。
X-linked hyper-IgM syndrome (XHIM) is a primary immunodeficiency due to mutations in CD40 ligand that affect immunoglobulin class switch recombination and somatic hypermutation. The disease is amenable to gene therapy using retroviral vectors, but dysregulated gene expression resulted in abnormal lymphoproliferation in mouse models, highlighting the need for alternative strategies. Here, we demonstrate the ability of both the TALEN and CRISPR/Cas9 platforms to efficiently drive integration of a normal copy of the CD40L cDNA delivered by Adeno-Associated Virus. Site-specific insertion of the donor sequence downstream of the endogenous CD40L promoter maintained physiologic expression of CD40L while overriding all reported downstream mutations. High levels of gene modification were achieved in primary human hematopoietic stem cells (HSC) as well as in cell lines and XHIM patient-derived T cells. Notably, gene corrected HSC engrafted in immunodeficient mice at clinically-relevant frequencies. These studies provide the foundation for a permanent curative therapy in XHIM.
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