Efficient modification of CCR5 in primary human hematopoietic cells using a megaTAL nuclease and AAV donor template.
Efficient modification of CCR5 in primary human hematopoietic cells using a megaTAL nuclease and AAV donor template.
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DOI：
10.1126/scitranslmed.aac5530
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发表时间：
2015-09-30
影响因子：
17.1
通讯作者：
Rawlings DJ
中科院分区：
文献类型：
--
作者：
Sather BD;Romano Ibarra GS;Sommer K;Curinga G;Hale M;Khan IF;Singh S;Song Y;Gwiazda K;Sahni J;Jarjour J;Astrakhan A;Wagner TA;Scharenberg AM;Rawlings DJ
破坏HIV共同受体CCR5的基因突变或工程核酸酶可以阻止HIV对CD4+T细胞的感染。这些发现推动了CCR5特异性核酸酶的工程应用于艾滋病毒治疗。这种方法的有效性依赖于CCR5的有效双等位基因中断，而有效靶向赋予HIV对CCR5基因的耐药性的序列的能力有可能进一步改善临床结果。我们使用基于RNA的核酸酶表达和腺相关病毒(AAV)介导的CCR5靶向供体模板传递来在原代人类T细胞中实现高效的靶向重组。这种方法在T细胞中持续获得8%到60%的同源定向重组率，其中80%以上的细胞用MND-GFP表达盒修饰后表现出双等位基因修饰。MND-GFP修饰的T细胞保持着多样化的谱系，并与未修饰的细胞一样有效地植入免疫缺陷小鼠体内。利用这种方法，我们将编码嵌合抗原受体(CARS)的序列整合到CCR5基因座上，得到的靶向CAR T细胞具有抗肿瘤或抗HIV活性。或者，我们引入了C46 HIV融合抑制剂，产生具有高双等位基因CCR5破坏的T细胞群体，并通过CXCR4共受体取向对HIV具有潜在的保护作用。最后，将该方法应用于成人动员的CD34+细胞，获得了15%到20%的同源基因靶向。我们的结果表明，在原代人类造血细胞中进行高效的靶向整合是可行的，并突出了基因编辑在设计具有多种功能特性的T细胞产品方面的潜力。
Genetic mutations or engineered nucleases that disrupt the HIV co-receptor CCR5 block HIV infection of CD4+ T cells. These findings have motivated the engineering of CCR5-specific nucleases for application as HIV therapies. The efficacy of this approach relies on efficient biallelic disruption of CCR5, and the ability to efficiently target sequences that confer HIV resistance to the CCR5 locus has the potential to further improve clinical outcomes. We used RNA-based nuclease expression paired with adeno-associated virus (AAV) – mediated delivery of a CCR5-targeting donor template to achieve highly efficient targeted recombination in primary human T cells. This method consistently achieved 8 to 60% rates of homology-directed recombination into the CCR5 locus in T cells, with over 80% of cells modified with an MND-GFP expression cassette exhibiting biallelic modification. MND-GFP – modified T cells maintained a diverse repertoire and engrafted in immune-deficient mice as efficiently as unmodified cells. Using this method, we integrated sequences coding chimeric antigen receptors (CARs) into the CCR5 locus, and the resulting targeted CAR T cells exhibited antitumor or anti-HIV activity. Alternatively, we introduced the C46 HIV fusion inhibitor, generating T cell populations with high rates of biallelic CCR5 disruption paired with potential protection from HIV with CXCR4 co-receptor tropism. Finally, this protocol was applied to adult human mobilized CD34+ cells, resulting in 15 to 20% homologous gene targeting. Our results demonstrate that high-efficiency targeted integration is feasible in primary human hematopoietic cells and highlight the potential of gene editing to engineer T cell products with myriad functional properties.