CAREER: Extracellular Barriers to Adeno-Associated Viral Gene Therapy

基因治疗提供了潜在地治愈或停止具有已知遗传起源（例如囊性纤维化）或具有已知遗传修饰物（例如癌症）的疾病的进展的手段。基于病毒的方法主要用于基因治疗应用，因为它们具有将遗传货物有效递送至靶组织的天然能力。腺相关病毒（AAV）已经成为一种领先的治疗性基因递送系统，并且最近成为第一种被美国食品和药物管理局（FDA）批准用于临床的病毒。鉴于其日益增长和广泛的潜在用途，重要的是要了解AAV如何在整个身体中运输和分布，以确保它是基因治疗应用的安全有效的模式。先前的工作已经表明，AAV与血流和组织（如脑、肝和心脏）的组分的相互作用可以导致AAV在靶器官内的失活或限制其渗透。这个NSF CAREER项目的目的是开发必要的工具来全面评估AAV基因治疗的这些生物学障碍，以优化其性能并最大限度地提高关节炎，癌症和血友病等疾病的治疗效益。该项目包括一个协同教育计划，促进研究曝光和URM学生的职业机会的认识，提高本科课程，并提供培训机会，在研究本科生，研究生和博士后研究人员来自不同背景。 具体活动包括为巴尔的摩市的科学教师开发夏季研究沉浸和教育实验室活动开发计划，并将新的实验室模块集中在血液中的纳米和微粒运输到生物流体的本科课程中。 该CAREER项目的长期目标是建立定量工具，以支持使用天然和新型生物工程AAV的基因疗法的开发和合理设计。 虽然AAV显示与血液中血清蛋白的依赖型相互作用，其可以抑制或增强AAV介导的基因转移，但尚不清楚这些与血清蛋白的相互作用是否促进或损害AAV在循环中的稳定性。此外，许多AAV血清型用于细胞进入的靶受体（例如硫酸乙酰肝素）以高水平存在于细胞外基质（ECM）中，这可能导致AAV粘附于ECM，并因此导致在靶组织中的不良分布。然而，这些潜在的障碍，有效的AAV基因治疗尚未研究。 为了解决这个问题，研究者将追求三个研究目标：（1）表征AAV-血清相互作用和蛋白质冠形成，（2）在生理流动条件下检查AAV在全血中的稳定性，以及（3）评估AAV通过ECM和离体组织的扩散。研究人员将利用专业知识测量和建模界面处的蛋白质吸附，蛋白质介导的颗粒聚集以及纳米级颗粒在3D生物基质中的扩散，以了解AAV在血液和组织微环境中的行为。这些目标的结果将用分析模型解释，以确定（1）血清蛋白的竞争性吸附和AAV颗粒上的蛋白质冠形成如何影响其在循环中的稳定性，以及（2）ECM的组织特异性性质（例如密度和组成）如何影响AAV在靶器官内的分布。如果成功，这项工作将建立新的检测方法，可推广到病毒基因治疗，并可用作临床前研究的额外筛选工具。该奖项反映了NSF的法定使命，并已被认为值得通过使用基金会的智力价值和更广泛的影响审查标准进行评估。

Gene therapy offers the means to potentially cure or halt the progression of diseases with known genetic origins (e.g. cystic fibrosis) or with known genetic modifiers (e.g. cancer). Virus-based approaches have been primarily used for gene therapy applications, as they possess the natural ability to efficiently deliver genetic cargo to target tissues. Adeno-associated virus (AAV) has emerged as a leading therapeutic gene delivery system and recently became the 1st virus to be granted approval by the Food and Drug Administration (FDA) for clinical use. Given its growing and broad potential utility, it is important to understand how AAV traffics and distributes throughout the body to ensure it is a safe and effective modality for gene therapy applications. Prior work has shown that the interactions of AAV with components of the bloodstream and tissues like the brain, liver, and heart can cause inactivation or limit penetration of AAV within target organs. The aim of this NSF CAREER project is to develop the tools necessary to comprehensively assess these biological barriers to AAV gene therapy in order to optimize its performance and maximize therapeutic benefits in diseases such as arthritis, cancer, and hemophilia. The project includes a synergistic education plan that promotes research exposure and awareness of career opportunities for URM students, enhances undergraduate courses and provides training opportunities in research for undergraduate, graduate, and postdoctoral researchers from diverse backgrounds. Specific activities include developing a summer research immersion and educational lab activity development program for science teachers in Baltimore City and incorporating new lab modules focused on nano- and microparticle transport in blood into an undergraduate course on biofluids. The long-term goal of this CAREER project is to build quantitative tools to support the development and rational design of gene therapies using natural and novel bioengineered AAVs. Though AAV shows serotype-dependent interactions with serum proteins in the blood that can either inhibit or enhance AAV-mediated gene transfer, it is unknown if these interactions with serum proteins promote or compromise the stability of AAV in circulation. In addition, target receptors used for cell entry by many AAV serotypes (e.g. heparan sulfate) are present at high levels in the extracellular matrix (ECM), which could lead to adhesion of AAV to the ECM and as a result, poor distribution in target tissues. However, these potential barriers to effective AAV gene therapy have not been studied. To address this issue, the investigator will pursue three research objectives: (1) characterize AAV-serum interactions and protein corona formation, (2) examine AAV stability in whole blood under physiological flow conditions, and (3) evaluate AAV diffusion through ECM and ex vivo tissues. The investigator will use expertise in measuring and modeling protein adsorption at interfaces, protein-mediated particle aggregation, and diffusion of nanoscale particles in 3D biological matrices to understand the behavior of AAV in the blood and tissue microenvironment. The results of these objectives will be interpreted with analytical models to determine (1) how competitive adsorption of serum proteins and protein corona formation on AAV particles influences their stability in circulation and (2) how tissue-specific properties of ECM (e.g. density and composition) influence distribution of AAV within target organs. If successful, this work will establish new assays that are generalizable to viral gene therapeutics and can be used as an additional screening tool for pre-clinical studies.This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.