Targeted Modification of Host and Proviral DNA to Treat Latent HIV Infection

描述（由申请人提供）：长期控制和治愈HIV的主要障碍是HIV在包含潜伏感染、静止和有效感染的CD4+ T细胞的储存库中持续存在。最近，一名HIV患者通过抗HIV细胞的造血细胞移植（HCT）治愈，证明了这一策略的巨大潜力。不幸的是，抗艾滋病毒的捐赠者极为罕见，严重限制了这种方法。在这里，我们提出了一个多研究者计划来研究患者和非人灵长类动物中HIV储存库的生物学，并制定新的策略来重新填充抗HIV细胞的造血系统。我们已经组建了一个由HIV、基因改造、转化研究和移植领域的领导者组成的团队。具体来说，我们将结合自体HCT和基因修饰方法，使我们能够破坏自体干细胞中的CCR5受体，并破坏这些细胞中包含的整合HIV前病毒。我们提出了5个高度集成的项目来追求我们的总体目标，并提出了5个核心来支持它们。项目1，造血细胞移植：清除潜伏HIV病毒库的平台（Ann Woolfrey， FHCRC），将寻求澄清清除潜伏HIV病毒库所需的制备方案和供体移植物的相对贡献。项目2，用于根除HIV的zfin修饰干细胞（Philip Gregory, Sangamo Biosciences），将寻求通过锌指核酸酶破坏CCR5位点，赋予移植受体自身的细胞稳定的抗感染能力。项目3，CCR5靶向控制非人类灵长类动物的HIV/SHIV (han - peter Kiem， FHCRC)，将使用非人类灵长类动物的HIV/SHIV感染模型来确定CCR5位点和HCT的基因修饰是否可以提供HIV的长期控制。项目4，利用工程归巢内切酶靶向破坏集成SHIV， （Keith Jerome， FHCRC），将寻求通过设计高度特异性的归巢内切酶来直接解决病毒持久性问题，该酶可以识别和突变感染猕猴造血细胞中的集成SHIV原病毒。项目5，锌指核酸酶和归巢内切酶mRNA和cDNA的Aptamer和Dendrimer递送（John Rossi, Beckman Research Institute of City of Hope），将开发和优化ZFNs和he递送到靶细胞的策略。鉴于我们在移植和HIV研究方面的领导作用，我们相信我们处于独特的地位，可以将这些概念从高度相关的非人灵长类HIV/SHIV模型转移到治愈HIV感染患者。

DESCRIPTION (provided by applicant): A major obstacle to long-term control and cure of HIV has been the persistence of HIV in reservoirs that contain latently infected, resting, and productively infected CD4+ T cells. The recent cure of a patient with HIV by hematopoietic cell transplantation (HCT) with HIV-resistant cells has demonstrated the enormous potential of this strategy. Unfortunately, HIV-resistant donors are extremely rare, severely limiting this approach. Here we propose a multi-investigator program to study the biology of the HIV reservoir in patients and nonhuman primates, and to develop novel strategies to repopulate the hematopoietic system with HIV-resistant cells. We have assembled a team consisting of leaders in the fields of HIV, genetic modification, translational research, and transplantation. Specifically, we will combine autologous HCT with genetic modification approaches, allowing us to disrupt the CCR5 receptor in autologous stem cells, and also destroy the integrated HIV provirus contained within those cells. We propose 5 highly integrated projects in pursuit of our overall goal, and 5 cores for their support. Project 1, Hematopoietic Cell Transplant: Platform for Purging the Latent HIV Reservoir (Ann Woolfrey, FHCRC), will seek to clarify the relative contributions of the preparative regimen and the donor graft required for purging the latent HIV reservoir. Project 2, ZFN-Modified Stem Cells for HIV Eradication (Philip Gregory, Sangamo Biosciences), will seek to endow the transplant recipient's own cells with stable resistance to infection, via zinc finger nuclease disruption of the CCR5 locus. Project 3, CCR5 Targeting to Control HIV/SHIV in Nonhuman Primates (Hans-Peter Kiem, FHCRC), will use a non-human primate model of HIV/SHIV infection to determine whether genetic modification of the CCR5 locus and HCT can provide long-term control of HIV. Project 4, Targeted Disruption of Integrated SHIV by Engineered Homing Endonucleases, (Keith Jerome, FHCRC), will seek to directly address the problem of viral persistence, by designing highly specific homing endonucleases that can recognize and mutate integrated SHIV provirus in infected macaque hematopoietic cells. Project 5, Aptamer and Dendrimer Delivery of Zn Finger Nuclease and Homing Endonuclease mRNA and cDNA (John Rossi, Beckman Research Institute of the City of Hope), will develop and optimize delivery strategies by which ZFNs and HEs can be delivered to their target cells. Given our leading roles in Transplantation and HIV research, we believe we are uniquely positioned to move these concepts from a highly relevant nonhuman primate HIV/SHIV model toward a cure for HIV-infected patients. PUBLIC HEALTH RELEVANCE: The long-term goal of this project is to combine our expertise in hematopoietic cell transplantation with our unique zinc finger nuclease and homing endonuclease technologies to specifically target and modify 1) the CCR5 co-receptor for HIV, and 2) the integrated provirus itself. This comprehensive plan offers a plausible pathway toward a cure for individuals infected with HIV. Project 1 - Hematopoietic Cell Transplant: Platform for Purging the Latent HIV Reservoir Project Leader (PL): Ann E. Woolfrey (Description as provided by applicant): Hematopoietic cell transplant (HCT) has been reported to have purged the reservoir of latently HIV-infected cells in one patient who received an HIV-resistant graft. For this approach to become broadly feasible, it is imperative that we determine the factors involved in HCT that contribute to eradication of the reservoir. Project 1 aims to determine what components of HCT are critical for elimination of the latent viral reservoir. The broad aim of Project 1 is to develop a platform for delivery of HIV-resistant cells to replace latently infected cells. In order to accomplish this broad goal, it is imperative that we determine the role of the separate mechanisms that may contribute to long-term elimination of the reservoir: 1) intensive chemo/radiotherapy which may eradicate recipient lymphocytes and dendritic or antigen presenting cells; and 2) reconstitution of immunity with HIV-uninfected and HIV-resistant cells. We hypothesize that both dose-intensity and graft source contribute separately to elimination of latently infected cells. In Specific Aim 1, we will investigate in human subjects both 1) the dose-intensity of conditioning that produces a meaningful reduction in the quantity of latent viral infection, and 2) the role of the graft source in eliminating latently infected cells. In Specific Aim 2, the effect of a lymphoablative conditioning on latently SHIV infection will be studied carefully in a macaque model, so as to separate the role of conditioning from the role of the graft source. Finally, we hypothesize that, in the absence of HIV-resistant cells, the latent reservoir ultimately will be replenished over time by low level replication of virus. In Specific Aim 3, we will investigate the source of HIV responsible for replenishing the latent reservoir, by a phylogenetic analysis of viral sequences obtained from blood, cerebral spinal fluid, and the intestinal tract, before and after HCT. Result from Project 1 will be used together with that from Project 5 to develop a clinical protocol for delivery of genetically modified HIV resistant cells. Project 1 will rely heavily on Core A and Core B, for support of the primate experiments. The phylogenetic sequencing and analysis will be performed by Core C. PUBLIC HEALTH RELEVANCE: To date, only one successful cure for HIV has been reported, that of an HIV-infected patient given an allogeneic hematopoietic cell transplant (HCT) from a CCR5n32 homozygous donor. The objective of Project 1 is to investigate HCT as a method to purge the latent HIV reservoir. Ultimately knowledge gained from Project 1 will be used to develop a platform for transplantation of HIV-resistant cells.