Development of Novel AIDS Gene Therapy Strategies in the Macaque SHIV Model

描述（由申请人提供）：本项目的目的是在临床相关的猕猴SHIV（猿猴人类免疫缺陷病毒）模型中开发和评价新的抗HIV基因治疗策略。高效抗逆转录病毒疗法（HAART）降低了与HIV感染相关的发病率和死亡率，但耐药病毒变体的出现、毒性和依从性问题是控制艾滋病的重大障碍。此外，最近有希望的疫苗试验未能防止艾滋病毒，强调了开发替代疗法的重要性。一种这样的替代疗法是对造血细胞进行遗传修饰，使其对HIV感染具有抗性或至少抑制HIV复制。在这里，我们将探讨两个主要策略来完成基因改造。我们将评估新的慢病毒和泡沫病毒载体，我们将研究靶向基因插入干扰CCR 5表达。我们建议的另一个关键方面是使用临床相关的非人灵长类艾滋病模型。我们最近表明，这两种载体系统都可以有效地将基因转移到非人灵长类动物的HSC中，并且我们有猕猴细胞中锌指介导的CCR 5破坏的初步数据。基于这些发现，我们提出了四个目标。在目标1中，我们将研究抑制HIV复制的组合转基因盒。我们将在慢病毒和泡沫病毒载体中研究这些盒，以确定这些载体系统首先在NOD/SCID小鼠中的安全性，然后在非人灵长类动物中的安全性。这些研究还将使我们能够确定泡沫病毒载体是否可以克服某些转基因盒的慢病毒载体对载体滴度的不利影响。我们建议测试的抗HIV载体含有MGMT转基因，以允许移植后的体内选择。这些研究将使我们能够比较基因标记，植入和体内选择与这两个有前途的载体系统。在目标2中，我们将建立有效的基因修饰细胞的体内选择条件，并探索降低整合载体前病毒激活增强子的可能性的方法。在目标3中，我们将挑战具有高标记水平的猴子，以确定我们的基因治疗方法是否可以在体内保护免受SHIV感染。在目标4中，我们将开发使用靶向基因插入在确定的染色体位点引入抗HIV基因的方法，以避免载体介导的白血病发生的可能性。这种方法具有额外的优点，即CCR 5的敲除和抗HIV转基因的插入都应该增加基因修饰细胞对SHIV感染的抗性。SHIV含有几个HIV基因，包括达特，rev和env，我们将在我们的macaque-SHIV模型中靶向抑制病毒复制。拟议的研究将使我们能够在高度相关的猴子模型中测试基因治疗方法，并开发将治疗性转基因有效安全地递送至HSC和T细胞的方法。由于我们开发的载体含有在SHIV猴模型和人类HIV感染中发挥作用的抗HIV转基因，因此拟议的研究还应为未来的临床试验提供有希望的载体的关键疗效和安全性数据。公共卫生相关性：这项研究将使用高度临床相关的猴子模型来评估新型基因治疗策略治疗艾滋病的有效性和安全性。如果成功的话，这些研究将为使用干细胞基因疗法保护个人免受艾滋病感染带来新的方法。

DESCRIPTION (provided by applicant): The objective of this project is to develop and evaluate novel anti-HIV gene therapy strategies for AIDS in a clinically relevant macaque SHIV (simian human immunodeficiency viruses) model. Highly active antiretroviral therapy (HAART) has reduced the morbidity and mortality associated with HIV infection, but the emergence of resistant viral variants, toxicity, and compliance issues are significant obstacles in controlling AIDS. Additionally, recent promising vaccine trials have failed to protect from HIV, emphasizing the importance of developing alternative therapies. One such alternative therapy is the genetic modification of hematopoietic cells to make them resistant to HIV infection or to at least inhibit HIV replication. Here we will explore two main strategies to accomplish the genetic modification. We will evaluate novel lenti and foamy virus vectors and we will study targeted gene insertion to interfere with CCR5 expression. Another key aspect of our proposal is the use of a clinically relevant nonhuman primate AIDS model. We have recently shown that both vector systems allow for efficient gene transfer to HSCs in nonhuman primates, and we have preliminary data of zinc finger mediated CCR5 disruption in macaque cells. Based on these findings we propose 4 aims. In aim 1 we will study combinatorial transgene cassettes that inhibit HIV replication. We will study these cassettes in lentivirus and foamy virus vectors to determine the safety of these vectors systems first in NOD/SCID mice and then in the nonhuman primate setting. These studies will also allow us to determine whether foamy virus vectors can overcome the adverse effect on vector titers with lentivirus vectors for some transgene cassettes. The anti-HIV vectors we propose to test contain MGMT transgenes to allow in vivo selection post-transplantation. These studies will allow us to compare gene marking, engraftment and in vivo selection with these two promising vector systems. In aim 2 we will establish conditions for efficient in vivo selection of gene-modified cells and explore means to reduce the potential for enhancer activation by integrated vector proviruses. In aim 3 we will challenge monkeys that have high marking levels to determine if our gene therapy approach can protect from SHIV infection in vivo. In aim 4 we will develop methods to introduce anti-HIV genes at a defined chromosomal locus using targeted gene insertion in order to avoid the potential for vector-mediated leukemogenesis. This approach has the added advantage that knockout of CCR5 and insertion of an anti-HIV transgene should both increase the resistance of gene-modified cells to SHIV infection. SHIVs contain several HIV genes including tat, rev and env that we will target to inhibit viral replication in our macaque-SHIV model. The proposed studies will allow us to test the gene therapy approach in a highly relevant monkey model and to develop methods for efficient and safe delivery of therapeutic transgenes to HSCs and to T cells. Since the vectors we have developed contain anti-HIV transgenes that function both in the SHIV monkey model and for HIV infection in humans, the proposed studies should thus also provide critical efficacy and safety data for promising vectors for future clinical trials. PUBLIC HEALTH RELEVANCE: The proposed research will evaluate the efficacy and safety of novel gene therapy strategies for the treatment of AIDS using a highly clinically relevant monkey model. If successful the proposed studies will lead to novel approaches to protect individuals from AIDS using stem cell gene therapy.