Department of Molecular and Experimental Medicine, The Scripps Research Institute, La Jolla, California 92037, USA.
Mol Ther. 2012 Apr;20(4):849-59. doi: 10.1038/mt.2011.310. Epub 2012 Jan 24.
HIV-1-infected individuals can harbor viral isolates that can use CCR5, as well as CXCR4, for viral entry. To genetically engineer HIV-1 resistance in CD4(+) T cells, we assessed whether transient, adenovirus delivered zinc-finger nuclease (ZFN) disruption of genomic cxcr4 or stable lentiviral expression of short hairpin RNAs (shRNAs) targeting CXCR4 mRNAs provides durable resistance to HIV-1 challenge. ZFN-modification of cxcr4 in CD4(+) T cells was found to be superior to cell integrated lentivirus-expressing CXCR4 targeting shRNAs when CD4(+) T cells were challenged with HIV-1s that utilizes CXCR4 for entry. Cxcr4 disruption in CD4(+) T cells was found to be stable, conferred resistance, and provided for continued cell enrichment during HIV-1 infection in tissue culture and, in vivo, in peripheral blood mononuclear cell transplanted NSG mice. Moreover, HIV-1-infected mice with engrafted cxcr4 ZFN-modified CD4(+) T cells demonstrated lower viral levels in contrast to mice engrafted with unmodified CD4(+) T cells. These findings provide evidence that ZFN-mediated disruption of cxcr4 provides a selective advantage to CD4(+) T cells during HIV-1 infection.
HIV-1 感染个体可以携带能够利用 CCR5 和 CXCR4 进行病毒进入的病毒分离株。为了在 CD4(+)T 细胞中遗传工程改造 HIV-1 抗性，我们评估了瞬时腺病毒递送锌指核酸酶 (ZFN) 破坏基因组 cxcr4 或稳定慢病毒表达靶向 CXCR4 mRNAs 的短发夹 RNA (shRNA) 是否为 HIV-1 挑战提供持久的抗性。当 CD4(+)T 细胞受到利用 CXCR4 进入的 HIV-1 攻击时，发现 ZFN 修饰的 cxcr4 在 CD4(+)T 细胞中优于细胞整合的慢病毒表达靶向 CXCR4 的 shRNA。发现 CD4(+)T 细胞中的 cxcr4 破坏是稳定的，赋予了抗性，并在组织培养中的 HIV-1 感染期间和体内移植 NSG 小鼠的外周血单核细胞中持续进行细胞富集。此外，与移植未修饰的 CD4(+)T 细胞的小鼠相比，移植了 cxcr4 ZFN 修饰的 CD4(+)T 细胞的 HIV-1 感染小鼠的病毒水平较低。这些发现提供了证据，证明 ZFN 介导的 cxcr4 破坏在 HIV-1 感染期间为 CD4(+)T 细胞提供了选择性优势。
