Nanoscience-Inspired Acoustofluidic Assembly Lines for Gene and Cellular Therapies

项目摘要 基于干细胞的基因疗法，利用基因编辑方法来解决致病突变 正在成为各种病理的可行医疗干预措施。目前基于病毒载体的 递送基因编辑机制的非病毒基因转移方法，其涉及化学或能量 细胞膜的破坏，在实验室环境中常规使用，但在临床上按比例扩大时， 相关应用，其目标是制造治疗性细胞产品。新的方法， 需要快速、安全和经济地交付基因编辑包，以支持 需要将这些基因疗法广泛地应用于患者护理。我们的解决方案， 未满足的需求利用基因编辑和纳米技术的创新，使细胞膜瞬时 多孔的，使得能够在细胞内递送生物分子货物。我们将设计和应用新的方法， 在微流体系统内产生的声波（即，声流体学）以机械地破坏细胞 膜，促进CRISPR/Cas9基因编辑组件的快速有效递送， 包装在超分子纳米载体中。我们使用镰状细胞病，一种最常见的 全球范围内的血红蛋白病，作为评价拟议平台的初始临床目标，因为它源于 一种明确定义的基因突变，可以靶向造血干细胞中的位点特异性校正， 基因编辑系统，如CRISPR/Cas9。这项研究的成功实施将为以下方面铺平道路： 能够在临床上快速和可持续地处理基于干细胞的基因疗法的技术- 适用剂量-有效地建立可扩展的，良好的生产规范兼容的装配线， 制造基因修饰的治疗性细胞产品以治疗多种疾病， 未来细胞疗法的临床应用

Project Summary Stem cell-based gene therapies that leverage gene-editing approaches to address disease-causing mutations are emerging as viable medical interventions across a variety of pathologies. Current viral-vector-based and non-viral gene-transfer methods of delivering gene editing machinery, which involve either chemical or energetic disruption of cell membranes, are used routinely in laboratory settings, but fall short when scaled up for clinically relevant applications targetting the manufacture of therapeutic cell products. New methods that enable efficient, rapid, safe, and economical delivery of gene editing packages are needed to support the infrastructures that will be required to translate these gene therapies broadly for application in patient care. Our solution to this critical unmet need leverages innovations in gene editing and nanotechnology to render cell membranes transiently porous, enabling intracellular delivery of biomolecular cargoes. We will design and apply new methods that use acoustic waves generated within microfluidic systems (i.e., acoustofluidics) to mechanically disrupt cell membranes, facilitating the rapid and efficient delivery of CRISPR/Cas9 gene-editing components that are packaged into supramolecular nanocarriers. We use sickle cell disease, one of the most common hemoglobinopathies worldwide, as an initial clinical target for evaluating the proposed platform as it arises from a well-defined genetic mutation that can be targetted for site-specific correction in hematopoietic stem cells with gene editing systems such as CRISPR/Cas9. Successful execution of this research will pave the way for technologies that enable rapid and sustainable processing of stem cell-based gene therapies at clinically- applicable doses – effectively establishing scalable, good manufacturing practice-compatible assembly lines for manufacturing gene modified therapeutic cell products to treat a wide variety of disesases and will streamline the clinical deployment of future cellular therapies.