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

项目总结/摘要 心血管疾病是美国人死亡的主要原因。基因治疗在乳腺癌中的应用研究 心血管目的正在迅速发展，但面临着关键问题，如脱靶效应和宿主 免疫系统对病毒载体的反应腺相关病毒（AAV）是一种很有前途的基因递送载体， 已在全球200多个临床试验中使用。该项目的目标是创建一个改进的AAV- 基于载体的靶向递送，并表征宿主对该载体的免疫应答， 到未修饰的AAV衣壳。我们的实验室已经创建了一个蛋白酶激活的AAV载体，称为provector， 可以将转基因传递到细胞外蛋白酶升高的位点。provector被锁定（“OFF”状态），直到 被基质金属蛋白酶切割（“ON”状态），其在疾病如心肌梗死后 心肌梗死、心脏重塑、充血性心力衰竭和动脉粥样硬化。然而，原型provector， 具有不完全的去靶向，这可能导致不期望的副作用。该项目旨在优化 前载体在关闭状态下去靶向，同时保持转导在开启状态。此外，体内 行为的优化provector将探讨和关键组成部分的免疫反应， 将鉴定前载体和未修饰的衣壳。 为了实现这些目标，将完成两个具体目标。首先，将对provector进行优化， 增加的靶向激活和减少的脱靶活性。为此，将在 其中一些亚基将含有为在OFF状态下降低转导而优化的强肽锁， 而其它的将是野生型以增加ON状态活性。病毒的物理特性和ON 和OFF转导谱将在体外表征。第二，免疫反应中的关键成分 将对AAV进行鉴定。将施用优化的前载体和野生型AAV衣壳 免疫活性和几种免疫缺陷小鼠品系。病毒的整体行为和免疫 将在一次给药后和再给药后测量产生的反应。的答复 将比较不同的小鼠品系，以鉴定必需的免疫成分及其对病毒的影响。 行为和免疫反应。这项研究意义重大，因为它将创造一种更有效的蛋白酶- 可激活的前载体，并将提供各种免疫系统成分的效果的比较研究 对抗AAV免疫应答和病毒行为的影响。因此，本研究将确定能够 成为未来开发免疫逃避载体的目标。这些结果具有显著的 对临床使用的靶向和免疫逃避载体的发展的影响，这将大大 推进基于AAV的基因治疗领域，并为各种疾病创造新的治疗方法。

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.