Gene-Editing of Human Pluripotent Stem Cell-Derived Cardiovascular Therapy Grafts to Improve Transplant Outcomes

摘要 首次心肌梗死后，36%的男性患者和47%的女性患者在5年内死亡。这 说明了当前治疗干预措施的不足。我们实验室的长期目标是 开发基于修复性多能干细胞(PSC)的免疫耐受且有意义的疗法 改善患者健康和生活质量。此R01应用程序的总体目标是：1)使用 CRISPR/Cas9基因编辑技术用于靶向人PSC表面的黏附分子 心血管治疗(PSC-CVT)破坏血管化血管的黏附、渗透和破坏 通过同种异体免疫细胞进行移植；以及2)确定最佳细胞组成和免疫原性 新一代低免疫PSC-CVT移植物在炎症环境中最大限度地发挥修复能力 密西西比州。我们的中心假设是，定向删除AM基因将促进PSC的免疫耐受。 CVTS通过两种机制：1)减少免疫细胞接触介导的破坏；2)抗炎 与基因干扰AM直接相关的效应(例如，分泌因子和基因表达的变化) 功能。这个项目的基本原理是免疫低下的pscs将成为未来的主要临床平台。 需要多年的时间和改进的基因编辑方法来实现PSC移植物的有效免疫耐受。 此外，本项目中AM基因编辑的成功验证将提供一种新的推进途径 其他疾病的未来移植疗法。为了达到我们的目标，我们将实现以下目标 特异性靶点(SA1)：确定AM消融对免疫细胞接触介导的PSC-CVT移植物的影响 破坏；SA2)定义由免疫细胞启动的炎症反应：PSC-CVT移植物的相互作用；以及 SA3)检测AM基因敲除的PSC-CVT的体内修复能力和免疫耐受潜能 炎症性心肌梗死环境中的移植物。这项研究具有重要意义，因为它验证了一种新的贪污策略和 低免疫PSC疗法测试平台，具有拯救生命和提高患者生活质量的巨大潜力 许多MI患者和其他以细胞免疫功能障碍为特征的病理改变 解剖位置。它的创新之处在于：1)针对免疫细胞黏附的新方法 预计将阻止适应性和先天免疫细胞介导的移植物破坏的方式；2)它使用三种 经优化的细胞PSC-CVT移植物具有优越的修复功能和低免疫原性； 使用我们实验室开发的先进分析和模型严格询问人类的免疫反应。 最终，这项工作将开发出一种突破性的心脏疗法，非常适合临床试验，具有潜在的 拯救生命，提高数百万患者的生活质量。

ABSTRACT After a first myocardial infarction (MI), 36% of male and 47% of female patients die within 5 years. This illustrates the inadequacy of current therapeutic interventions. The long-term goal of our laboratory is to develop reparative pluripotent stem cell (PSC)-based therapies that are immune-tolerated and meaningfully improve patient health and quality of life. The overall objectives of this R01 application are to: 1) use CRISPR/Cas9 gene-editing approaches to target adhesion molecules (AMs) on human PSC-derived cardiovascular therapies (PSC-CVTs) to disrupt the adherence, infiltration, and destruction of vascularized grafts by allogeneic immune cells; and 2) define optimal cellular composition and immunogenicity profiles of next-generation hypoimmune PSC-CVT grafts to maximize their reparative capacity in the inflammatory setting of MI. Our central hypothesis is that targeted deletion of AM genes will facilitate immune tolerance of PSC- CVTs via two mechanisms: 1) diminished immune cell contact-mediated destruction; and 2) anti-inflammatory effects (e.g., secreted factor and gene expression changes) directly associated with genetically disrupting AM function. The rationale for this project is that hypoimmune PSCs will be key clinical platforms in the coming years and improved gene-editing approaches are needed to achieve effective immune tolerance of PSC grafts. Additionally, successful validation of AM gene-editing in this project will provide a new avenue for advancing future transplantation therapies for other diseases. To attain our objectives, we will pursue the following specific aims (SAs): SA1) Define the effects of AM ablation on immune cell contact-mediated PSC-CVT graft destruction; SA2) Define the inflammatory responses initiated by immune cell:PSC-CVT graft interactions; and SA3) Determine the in vivo reparative capacity and immune-tolerance potential of AM knockout PSC-CVT grafts in the inflammatory MI setting. This research is significant because it validates a new graft strategy and testing platform for hypoimmune PSC therapies, with great potential to save lives and improve quality of life for many patients with MI and other pathologies characterized by cellular dysfunction in immune-competent anatomical sites. It is innovative because: 1) of the new approach of targeting immune cell adhesion in a manner anticipated to impede both adaptive and innate immune cell-mediated graft destruction; 2) it uses a tri- cellular PSC-CVT graft optimized for superior reparative function and hypoimmunogenicity; and 3) we rigorously interrogate the human immune response using advanced assays and models developed in our lab. Ultimately, this work will develop a breakthrough cardiac therapy well-suited for clinical trials, with the potential to save lives and improve the quality-of-life for millions of patients.