ENGINEERING HIV-RESISTANT HUMAN CD4+ T CELLS

这个子项目是许多利用资源的研究子项目之一 由NIH/NCRR资助的中心拨款提供。子项目的主要支持 而子项目的主要调查员可能是由其他来源提供的， 包括其它NIH来源。 列出的子项目总成本可能 代表子项目使用的中心基础设施的估计数量， 而不是由NCRR赠款提供给子项目或子项目工作人员的直接资金。 HIV-1进入需要宿主细胞上的CD 4和CCR 5或CXCR 4共受体的细胞表面表达。CCR 5 <$32多态性纯合子个体不表达CCR 5，并保护其免受CCR 5嗜性（R5）病毒株的感染。作为灭活CCR 5的方法，我们在过继转移之前将CCR 5特异性锌指核酸酶引入人CD 4 + T细胞中，但是保护细胞免受使用CXCR 4代替（X4）或除了CCR 5之外使用（R5 X4）的病毒株的需要仍然是一个问题。在这里，我们描述了一对锌指核酸酶的工程，当引入到人类T细胞，有效地破坏CXCR 4的切割和易错的非同源DNA末端连接。所得到的细胞正常增殖，并且对使用X4-的HIV-1菌株的感染具有抗性。CXCR 4也可以在CCR 5 <$32 CD 4 + T细胞中失活，我们表明这些细胞对所有测试的HIV-1菌株都有抗性。在人源化小鼠模型中，CXCR 4的缺失也提供了对X4 HIV-1的保护，尽管这种保护随着时间的推移由于使用R5的病毒突变体的出现而丧失。这些数据表明，CXCR 4-特异性ZFN可能被证明是有用的，在建立对CXCR 4-嗜性HIV的抗性，用于HIV感染个体的自体移植。

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. Primary support for the subproject and the subproject's principal investigator may have been provided by other sources, including other NIH sources. The Total Cost listed for the subproject likely represents the estimated amount of Center infrastructure utilized by the subproject, not direct funding provided by the NCRR grant to the subproject or subproject staff. HIV-1 entry requires the cell surface expression of CD4 and either the CCR5 or CXCR4 coreceptors on host cells. Individuals homozygous for the CCR5¿32 polymorphism do not express CCR5 and are protected from infection by CCR5-tropic (R5) virus strains. As an approach to inactivating CCR5, we introduced CCR5 specific zinc-finger nucleases into human CD4+ T cells prior to adoptive transfer, but the need to protect cells from virus strains that use CXCR4 in place of (X4) or in addition to CCR5 (R5X4) remains a concern. Here we describe engineering a pair of zinc finger nucleases that, when introduced into human T cells, efficiently disrupt CXCR4 by cleavage and error-prone nonhomologous DNA end-joining. The resulting cells proliferated normally and were resistant to infection by X4-using HIV-1 strains. CXCR4 could also be inactivated in CCR5¿32 CD4+ T cells, and we show that such cells were resistant to all strains of HIV-1 tested. Loss of CXCR4 also provided protection from X4 HIV-1 in a humanized mouse model, though this protection was lost over time due to the emergence of R5-using viral mutants. These data suggest that CXCR4-specific ZFNs may prove useful in establishing resistance to CXCR4-tropic HIV for autologous transplant in HIV-infected individuals.