Division of Allergy & Immunology, Department of Pediatrics, David Geffen School of Medicine at the 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.
Cell Rep. 2018 May 29;23(9):2606-2616. doi: 10.1016/j.celrep.2018.04.103.
X-linked hyper-immunoglobulin M (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 results in abnormal lymphoproliferation in mouse models, highlighting the need for alternative strategies. Here, we demonstrate the ability of both the transcription activator-like effector nuclease (TALEN) and clustered regularly interspaced short palindromic repeats-associated protein 9 (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 (HSCs), as well as in cell lines and XHIM-patient-derived T cells. Notably, gene-corrected HSCs engrafted in immunodeficient mice at clinically relevant frequencies. These studies provide the foundation for a permanent curative therapy in XHIM.
X 连锁高免疫球蛋白 M（hyper-IgM）综合征（XHIM）是一种由 CD40 配体突变引起的原发性免疫缺陷病，影响免疫球蛋白类别转换重组和体细胞超突变。该疾病可通过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异常的淋巴增殖，这突出表明需要替代策略。在这里，我们证明了转录激活因子样效应物核酸酶（TALEN）和成簇规律间隔短回文重复相关蛋白 9（CRISPR/Cas9）平台都能够有效地驱动腺相关病毒（Adeno-Associated Virus）递送的正常 CD40L cDNA 的整合。供体序列在内源性 CD40L 启动子下游的特异性插入维持了 CD40L 的生理表达，同时覆盖了所有报道的下游突变。在原代人类造血干细胞（HSCs）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 HSCs 以临床相关频率植入免疫缺陷小鼠中。这些研究为 XHIM 的永久性治愈疗法奠定了基础。
