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

摘要 在首次心肌梗死（MI）后，36%的男性和47%的女性患者在5年内死亡。这 说明了目前治疗干预的不足。我们实验室的长期目标是 开发基于修复性多能干细胞（PSC）的疗法，这些疗法具有免疫耐受性， 改善患者健康和生活质量。本R 01应用程序的总体目标是：1）使用 CRISPR/Cas9基因编辑方法靶向人PSC衍生的粘附分子（AM） 心血管治疗（PSC-CVTs），以破坏血管化的粘附、浸润和破坏。 同种异体免疫细胞的移植物;和2）确定最佳的细胞组成和免疫原性概况， 下一代低免疫PSC-CVT移植物，以最大限度地提高其在炎症环境中的修复能力 的MI。我们的中心假设是AM基因的靶向缺失将促进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 效果（例如，分泌因子和基因表达变化）直接与遗传破坏AM 功能该项目的基本原理是低免疫性PSC将成为未来的关键临床平台 需要多年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，该项目中AM基因编辑的成功验证将为推进 未来的移植治疗其他疾病。为达致这些目标，我们会致力 特定目的（SA）：SA 1）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; SA 2）定义免疫细胞引发的炎症反应：PSC-CVT移植物相互作用;和 SA 3）确定AM敲除PSC-CVT的体内修复能力和免疫耐受潜力 移植物在炎症性心肌梗死中的作用这项研究意义重大，因为它验证了一种新的嫁接策略， 低免疫PSC疗法的测试平台，具有拯救生命和改善生活质量的巨大潜力， 许多患有MI和以免疫活性细胞中的细胞功能障碍为特征的其他病理的患者， 解剖部位。它是创新的，因为：1）靶向免疫细胞粘附的新方法， 预期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- 细胞PSC-CVT移植物经过优化，具有出色的上级修复功能和低免疫原性;和3）我们 使用我们实验室开发的先进分析和模型严格询问人类免疫反应。 最终，这项工作将开发出一种突破性的心脏治疗方法，非常适合临床试验，具有潜力。 拯救生命，改善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.