Efficient Modification of the CCR5 Locus in Primary Human T Cells With megaTAL Nuclease Establishes HIV-1 Resistance.
Efficient Modification of the CCR5 Locus in Primary Human T Cells With megaTAL Nuclease Establishes HIV-1 Resistance.
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DOI：
10.1038/mtna.2016.56
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发表时间：
2016-08-23
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HIV-1 辅助受体 CCR5 的 32 个碱基对自然缺失已被证明可以保护人类 CD4+ T 细胞免受 HIV 感染。最近的基因工程方法使用工程核酸酶来破坏基因并模拟这种突变，这为艾滋病毒治疗带来了希望。我们开发了一种针对 CCR5 第三个细胞外环的 megaTAL 核酸酶，通过 mRNA 转染将其递送至原代人 T 细胞。 CCR5 megaTAL 核酸酶在细胞系中建立了对 HIV 的抗性，并高效破坏原代人类 CD4+ T 细胞上的 CCR5 表达，通过分子分析测量，对原代细胞中的基因座进行了高达 80% 的修饰。当移植到免疫缺陷小鼠体内时，基因修饰细胞的移植水平与未修饰细胞相当。此外，在免疫缺陷小鼠模型中，在体内 HIV-1 感染期间，基因修饰的 CD4+ 细胞优先扩增。我们的结果证明了使用工程化的megaTAL核酸酶靶向原代T细胞中的CCR5的可行性，以及使用基因修饰细胞重建患者免疫系统并提供针对HIV感染的保护的潜力。
A naturally occurring 32-base pair deletion of the HIV-1 co-receptor CCR5 has demonstrated protection against HIV infection of human CD4+ T cells. Recent genetic engineering approaches using engineered nucleases to disrupt the gene and mimic this mutation show promise for HIV therapy. We developed a megaTAL nuclease targeting the third extracellular loop of CCR5 that we delivered to primary human T cells by mRNA transfection. The CCR5 megaTAL nuclease established resistance to HIV in cell lines and disrupted the expression of CCR5 on primary human CD4+ T cells with a high efficiency, achieving up to 80% modification of the locus in primary cells as measured by molecular analysis. Gene-modified cells engrafted at levels equivalent to unmodified cells when transplanted into immunodeficient mice. Furthermore, genetically modified CD4+ cells were preferentially expanded during HIV-1 infection in vivo in an immunodeficient mouse model. Our results demonstrate the feasibility of targeting CCR5 in primary T cells using an engineered megaTAL nuclease, and the potential to use gene-modified cells to reconstitute a patient's immune system and provide protection from HIV infection.