Improved Zinc Finger Nuclease Delivery for HIV Gene Therapy

 描述(申请人提供)：锌指核酸酶(ZFN)介导的CCR5中断已被证明是HIV基因治疗的一种有前途的策略，但潜伏的病毒库仍然是绝育甚至是治愈HIV-1感染的主要障碍。静息的CD4+记忆T细胞被认为是潜伏期的主要来源，这些细胞也构成了一个特别稳定的病毒储存库。据报道，针对3‘和5’LTR保守序列的ZFN可以精确地切除J-Lat细胞中的整个前病毒，但将ZFN运送到静止的原代CD4+T细胞的困难限制了这种方法缩短潜伏期的潜力。最近，我们开发了一种利用HIV env伪分型非整合慢病毒的方法，成功地将CCR5-ZFN传递给静息的CD4+T细胞，并在Hu-PBL小鼠体内显示了有效性。我们还在初步研究中发现，使用Sindbis病毒糖蛋白工程表达ZZ结构域(来自蛋白A的Fc结合域)提供了另一种针对静止的CD4+T细胞的简单方法。在这里，假型慢病毒在感染T细胞之前用CD4抗体(包含慢病毒的ZZ结构域可以结合任何抗体)进行治疗。我们可以证明靶向作用于70%的静止的CD4+T细胞。因此，我们建议通过HIV包膜伪型或Sindbis病毒env-ZZ结构域，将携带LTR和/或CCR5 ZFN的非整合慢病毒靶向静止的CD4+T细胞，从而使基因编辑变得容易。我们将使用此方法编辑CCR5和/或HIV-LTR。因此，破坏旁观者CD4+T细胞中的CCR5将保护未感染的细胞，而LTR/ZFN将切除感染的原代CD4+T细胞中的整合前病毒。这两种战略的结合可能会提供完全的艾滋病毒保护。这项拟议研究的长期目标是开发一种替代的、有效的和安全的艾滋病毒疗法，这种疗法可能提供一种简化方案的功能性治愈。我们建议：1)制备和优化基于锌指核酸酶(ZFN)的静止CD4+T细胞基因编辑试剂；2)评估ART抑制个体的体外ZFN修饰的PBMC在重建HU/PBL小鼠后是否能防止内源性病毒的重新激活

 DESCRIPTION (provided by applicant): Zinc finger nucleases (ZFN)-mediated CCR5 disruption has shown to be a promising strategy for HIV gene therapy, but the latent viral reservoir still remains the major obstacle towards sterilizing or even a functional cure for HIV-1 infection. Resting CD4+ memory T cells are thought to be the major source of latency, and these also constitute a particularly stable reservoir of virus. ZFN targeting the conserved sequences in the 3' and the 5' LTR was reported to precisely excise the entire provirus in J-Lat cells, but the difficulty in delivering ZFNs to resting primary CD4+ T cells limited the potential f this approach for latency reduction. Recently, we have developed a method by using HIV env pseudotyping non-integrating lentivirus, successfully delivered CCR5-ZFN to resting CD4+ T cells and showed effectiveness in vivo in Hu-PBL mice. We have also found in preliminary studies that using Sindbis virus glycoprotein engineered to express ZZ-domain (Fc binding domain from protein A) provides another easy way to target resting CD4+ T cells. Here the pseudotyped lentivirus is treated with CD4 antibody (the ZZ domain containing lentivirus can bind any antibody) before infecting T cells. We could demonstrate targeting in >70% of resting CD4+ T cells. Thus we propose to target resting CD4+ T cells with non-integrating lentivirus carrying LTR and/or CCR5 ZFNs via HIV env-pseudotyped or Sindbis virus env-ZZ-domain, by which it would enable easy gene editing. We will use this method to edit CCR5 and/or HIV-LTR. Therefore, CCR5 disruption in bystander CD4+ T cells would protect uninfected cells and LTR/ZFN would excise integrated provirus in infected primary CD4+ T cells. The combination of these two strategies would potentially provide complete HIV protection. The long-term goal of this proposed research is to develop an alternative effective and safe HIV therapy that might offer a functional cure with simplified regimen. We propose to: 1) Generate and optimize reagents for Zinc Finger Nuclease (ZFN)- based gene editing in resting CD4+ T cells; 2) Evaluate whether ex vivo ZFN-modified PBMCs from ART suppressed individuals are protected from reactivation of endogenous virus after reconstitution in Hu/PBL mice