Finger Nucleases to Specifically Disrupt Coreceptor Expression

耐药HIV变异体的出现构成了一个重大的临床挑战， 继续开发新的抗病毒药物，包括小分子CCR 5和CXCR 4拮抗剂。 最近，我们通过有针对性的永久破坏， 通过使用高度特异性的锌指核酸酶（ZFN）在人类T细胞中的ccr 5基因。在体外， ccr 5被破坏的细胞生长正常，对R5病毒株的感染有抗性，但对X4病毒株敏感 病毒株用R5-ZFN处理的CD 4 + T细胞移植并随后用R5-ZFN感染的NOG小鼠 病毒株表现出较低的病毒载量，并且具有破坏的ccr 5的细胞在 vivo.我们对项目3的假设是：1。原代CD 4 T细胞中CXCR 4和CCR 5的ZFN修饰 细胞将保护细胞免受HIV感染，同时限制NOG模型中X4菌株的遗传漂移。 收养转移，2.用CCRS修饰的CD 34+干细胞重建NOG小鼠将拓宽 对CCR 5-嗜性HIV的抗性，和3.用于过继转移的CD 4 T细胞中CCR 5的ZFN修饰 在对抑制剂耐药的情况下，比小分子抑制剂更具优势。 继续我们与Sangamo Biosciences的富有成效的合作，并通过迭代过程测试， 一组CXCR 4特异性ZFN（X4-ZFN），因为它们能够破坏编码这种重要HIV的基因， 共受体最有效和特异性地破坏CXCR 4的X4-ZFN（目标#1），部分确定为 通过与核心C和领先的CCR 5 ZFN合作，将测试其保护 细胞系和原代人T细胞在体外（目标#2）和体内被多种HIV-1毒株感染 与核心B合作使用NOG鼠标系统（目标#3）。我们还将结合我们的联合收割机的X4-和R5- ZFN试图使细胞完全抵抗HIV-1感染。虽然我们将在这两个方面瞄准CCR 5， 由于CXCR 4与T细胞和造血干细胞之间的相互作用，我们将CXCR 4的靶向限制在T细胞，因为CXCR 4具有其他靶向作用。 在造血中的重要功能（目标#2和#3）。因为基因切除病毒辅助受体 对病毒施加选择性压力，这与使用小分子辅助受体完全不同 拮抗剂，我们将监测我们的体外和体内感染实验的病毒耐药性的演变 (Aim#4），如果观察到，则表征机制。我们的长期目标是管理辅助受体缺陷 自体T细胞和干细胞用于HIV感染个体。

The emergence of drug-resistant HIV variants constitutes a significant clinical challenge that necessitates the continued development of new antiviral agents, including small molecule CCR5 and CXCR4 antagonists. Recently, we have conferred genetic resistance to HIV infection through the targeted, permanent disruption of the ccr5 gene in human T cells through the use of highly specific zinc-finger nucleases (ZFNs). In vitro, cells with disrupted ccr5 grow normally, are resistant to infection by R5 virus strains, but are sensitive to X4 virus strains. NOG mice engrafted with R5-ZFN treated CD4+ T cells and subsequently infected with an R5 virus strain exhibited lower virus loads, and cells with disrupted ccr5 had a significant survival advantage in vivo. Our hypotheses for Project 3 are: 1. ZFN modification of both CXCR4 and CCR5 in primary CD4 T cells will protect cells from HIV infection while limiting genetic drift toward X4 strains in the NOG model of adoptive transfer, 2. Reconstitution of NOG mice with CCRS-modified CD34+ stem cells will broaden resistance to CCR5-tropic HIV, and 3. ZFN modification of CCR5 in CD4 T cells for adoptive transfer is advantageous over small molecule inhibitors in the setting of resistance to inhibitor In this project, we will continue our productive collaboration with Sangamo Biosciences, and through an iterative process test a panel of CXCR4-specific ZFNs (X4-ZFN) for their ability to disrupt the gene encoding this significant HIV coreceptor. The X4-ZFNs that disrupt CXCR4 most efficiently and specifically (Aim #1), determined in part through working with the Core C, and the lead CCR5 ZFNs will be tested for the ability to confer protection of cell lines and primary human T cells to infection by a variety of HIV-1 strains in vitro (Aim #2), and in vivo using the NOG mouse system in collaboration with Core B (Aim #3). We will also combine our X4- and R5- ZFNs in an attempt to render cells completely resistant to HIV-1 infection. While we will target CCR5 in both T cells and hematopoietic stem cells, we will limit the targeting of CXCR4 to T cells since CXCR4 has other important functions in hematopoiesis (Aims #2 and #3). Because genetic ablation of viral coreceptors imposes a selective pressure on virus that is quite distinct from the use of small molecule coreceptor antagonists, we will monitor our in vitro and in vivo infection experiments for the evolution of viral resistance (Aim #4), and if observed characterize the mechanism(s). Our long-term goal is to administer coreceptordeficient autologous T cells and stem cells to HIV infected individuals.