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

描述(由申请人提供)：该项目的目标是在临床相关的猕猴SIV(猴人类免疫缺陷病毒)模型中开发和评估抗HIV基因治疗艾滋病的新策略。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，但耐药病毒变异体的出现、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预防艾滋病毒，这强调了开发替代疗法的重要性。其中一种替代疗法是对造血细胞进行基因改造，使其对艾滋病毒感染具有抵抗力，或者至少抑制艾滋病毒复制。在这里，我们将探索两种主要的策略来完成基因改造。我们将评估新的Lenti和泡沫病毒载体，并研究靶向基因插入来干扰CCR5的表达。我们建议的另一个关键方面是使用临床上相关的非人类灵长类艾滋病模型。我们最近已经证明，这两种载体系统都可以在非人类灵长类动物中有效地将基因转移到HSCs，我们已经有了锌指介导的CCR5在猕猴细胞中破坏的初步数据。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式磁带。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，然后在非人类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一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，以便在移植后进行体内选择。这些研究将使我们能够将基因标记、植入和体内选择与这两个有前景的载体系统进行比较。在目标2中，我们将建立有效的体内选择转基因细胞的条件，并探索通过整合载体前病毒降低增强子激活的可能性的方法。在目标3中，我们将挑战得分水平较高的猴子，以确定我们的基因治疗方法是否可以预防体内的希沃克病毒感染。在目标4中，我们将开发使用靶向基因插入在特定染色体位置引入抗HIV基因的方法，以避免载体介导的白血病发生的可能性。这种方法还有一个额外的优势，即敲除CCR5和插入抗HIV转基因都应该增加基因修饰细胞对Shiv感染的抵抗力。SHIV包含几个HIV基因，包括Tat、rev和env，我们将针对这些基因在我们的猕猴-SHIV模型中抑制病毒复制。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，并开发有效和安全地将治疗性转基因输送到HSCs和T细胞的方法。由于我们开发的载体含有在SIV猴模型和人类HIV感染中都起作用的抗HIV转基因，因此，拟议的研究也将为未来临床试验提供重要的有效性和安全性数据。公共卫生相关性：这项拟议的研究将使用高度临床相关的猴子模型来评估治疗艾滋病的新基因治疗策略的有效性和安全性。如果成功，拟议的研究将导致使用干细胞基因疗法保护个人免受艾滋病侵袭的新方法。

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.