Characterization of Immune Response to an Improved Matrix Metalloproteinase-Targeting Adeno-Associated Virus

项目摘要/摘要 在美国，心血管疾病是主要的死亡原因。基因治疗在癌症治疗中的应用研究进展 心血管目的正在迅速发展，但面临着一些关键问题，如偏离目标的效应和宿主 对病媒的免疫反应。腺相关病毒(AAV)是一种很有前途的基因递送载体 已在全球200多项临床试验中使用。该项目的目标是创建一种改进的AAV- 以靶向递送为基础的载体，并比较宿主对该载体的免疫应答 到未经修饰的AAV衣壳。我们的实验室已经创造了一种可被蛋白酶激活的AAV载体，称为前载体， 可以将转基因运送到胞外蛋白水解酶升高的部位。原向量被锁定(关闭状态)，直到 被基质金属蛋白酶分解(“开”状态)，这种酶在心肌梗死后等疾病中升高。 心肌梗死、心脏重构、充血性心力衰竭和动脉粥样硬化。然而，原型命题载体， 有不完全的去靶向，这可能会导致不希望看到的副作用。该项目旨在优化 PROVECTOR在OFF状态下去靶向，同时在ON状态下保持转导。此外，体内的 将探索优化的前导载体的行为以及免疫应答中的关键成分 原载体和未经修饰的衣壳将被鉴定。 为实现这些目标，将完成两个具体目标。首先，将对前导向量进行优化 增加了目标上的激活，减少了目标外的活动。为此，将在中创建镶嵌前向量 其中一些亚基将包含为在关闭状态下减少转导而优化的强肽锁， 而其他国家则将采取野性措施，以增加国家活动。病毒的物理特性和其他 而Off转导图谱将在体外进行表征。第二，免疫反应中的关键成分 将确定针对AAV的病毒。优化的前导载体和野生型AAV衣壳都将被注射 对免疫活性品系和几种免疫缺陷品系的小鼠。病毒的整体行为和免疫力 所产生的反应将在一次给药后和重新给药后进行测量。中国政府的回应 将对不同的小鼠品系进行比较，以确定必要的免疫成分及其对病毒的影响 行为和免疫反应。这项研究具有重要意义，因为它将产生一种更有效的蛋白酶- 并将提供对不同免疫系统组件影响的比较研究 关于抗AAV免疫反应和病毒行为的研究。因此，这项研究将确定可以 是未来免疫逃逸载体研究的重点。这些结果具有重要的意义 对临床使用的靶向和免疫规避载体的开发的影响，这将大大 推进以AAV为基础的基因治疗领域，创造多种疾病的新疗法。

PROJECT SUMMARY/ABSTRACT Cardiovascular disease is the leading cause of death in the US. Research on the use of gene therapy for cardiovascular purposes is quickly advancing but face critical problems, such as off-target effects and host immune response against the vectors. Adeno-associated virus (AAV) is a promising gene delivery vector and has been used in more than 200 clinical trials worldwide. The goal of this project is to create an improved AAV- based vector with targeted delivery and to characterize the host immune response to this vector in comparison to unmodified AAV capsid. Our lab has created a protease-activatable AAV vector, called the provector, that can deliver transgenes to sites with elevated extracellular proteases. The provector is locked (“OFF” state) until cleaved by matrix metalloproteinases (“ON” state), which are elevated in diseases such as post-myocardial infarction cardiac remodeling, congestive heart failure, and atherosclerosis. The prototype provector, however, has incomplete de-targeting, which may lead to undesired side-effects. This project seeks to optimize provector de-targeting in the OFF state while maintaining transduction in the ON state. Additionally, the in vivo behavior of the optimized provector will be explored and key components in the immune response to this provector and the unmodified capsid will be identified. To achieve these objectives, two specific aims will be completed. First, the provector will be optimized for increased on-target activation and decreased off-target activity. To do so, a mosaic provector will be created in which some subunits will contain a strong peptide lock optimized for decreased transduction in the OFF state, while others will be wild-type to increase ON state activity. The virus’s physical properties and ON and OFF transduction profiles will be characterized in vitro. Second, key components in the immune response against AAV will be identified. Both the optimized provector and the wild-type AAV capsid will be administered to immune competent and several immune deficient strains of mice. The overall virus behavior and immune response generated will be measured after one administration and after re-administration. The responses of different mice strains will be compared to identify essential immune components and their effect on virus behavior and immune response. This research is significant because it will create a more effective protease- activatable provector and will offer a comparative study on the effect of various immune system components on anti-AAV immune response and virus behavior. As a result, this study will identify key components that can be targeted for the developing of immune-evasive vectors in the future. These results have significant implications on the development of targeted and immune-evasive vectors for clinical use, which will greatly advance the field of AAV-based gene therapy and create new treatments for a variety of diseases.