Nanoscience-Inspired Acoustofluidic Assembly Lines for Gene and Cellular Therapies

项目摘要 利用基因编辑方法解决致病突变的基于干细胞的基因疗法 正在成为跨越各种病理的可行的医疗干预措施。目前以病毒载体为基础的 传递基因编辑机器的非病毒基因转移方法，涉及化学或能量 破坏细胞膜，在实验室环境中经常使用，但在扩大到临床应用时还不够 针对治疗性细胞产品的制造的相关应用。新方法，使高效、 需要快速、安全和经济地交付基因编辑包，以支持将 被要求广泛翻译这些基因疗法，以便在患者护理中应用。我们对这一关键问题的解决方案 未得到满足的需求利用基因编辑和纳米技术的创新来瞬间呈现细胞膜 多孔性，使生物分子货物能够在细胞内传递。我们将设计和应用新的方法，使用 在微流体系统(即声流体学)中产生的声波，以机械方式破坏细胞 膜，便于快速高效地输送CRISPR/Cas9基因编辑组件，这些组件是 被包装成超分子纳米载体。我们使用镰状细胞病，这是最常见的疾病之一 作为评估拟议平台的初始临床目标的全球血红蛋白病，因为它产生于 一种明确的基因突变，可以靶向于造血干细胞的定点纠正 基因编辑系统，如CRISPR/Cas9。这项研究的成功进行将为以下工作铺平道路 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- 适用的剂量-有效地建立可扩展的、符合良好制造实践的装配线 制造基因修饰的治疗性细胞产品来治疗各种疾病，并将简化 未来细胞疗法的临床部署。

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.