Development of a robust, vector-free intracellular delivery platform for immune cells

开发强大的、无载体的免疫细胞细胞内递送平台

• 批准号: 8906135
• 负责人: Katarina Blagovic
• 金额: $ 25.06万
• 依托单位: SQZ BIOTECHNOLOGIES COMPANY, INC.
• 依托单位国家: 美国
• 财政年份: 2015
• 资助国家: 美国
• 起止时间: 2015-04-01 至 2017-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8906135
• 关键词: Accounting; Address; Adverse effects; Antibodies; Antigen Presentation; Antigens; Buffers; Cell Death; Cell membrane; Cell physiology; Cells; Cytomegalovirus; Cytoplasm; Cytosol; Data; Dendritic Cells; Development; Device Designs; Devices; Dextrans; Disadvantaged; Disease; Electroporation; Engineering; Future; Geometry; Goals; Human; I-antigen; Immune; Immune response; Immune system; Immunologist; Immunology; Immunotherapy; In Vitro; Individual; Label; Length; Libraries; Liquid substance; Lymphocyte; Measures; Mechanics; Mediating; Membrane; Methods; Microfluidics; Modeling; Molecular; Mus; Nanostructures; Nucleic Acids; Ovalbumin; Pathway interactions; Peptides; Performance; Phenotype; Polysaccharides; Process; Property; Proteins; Protocols documentation; Recombinants; Research; Research Personnel; Rest; Site; Small Interfering RNA; Speed; System; T-Lymphocyte; Techniques; Technology; Therapeutic; Therapeutic Human Experimentation; TimeLine; Toxic effect; Translating; Width; Work; base; c-Myc Staining Method; combat; constriction; cytokine; design; genome integrity; induced pluripotent stem cell; insight; macromolecule; meetings; monocyte; nanoparticle; new technology; novel; prophylactic; public health relevance; research study; technology development; tool; transcription factor; uptake; vector

 DESCRIPTION (provided by applicant): During the past decades immunologists have deciphered many of the mechanisms by which the mammalian immune system either combats or mediates a host of diseases, and clinicians are beginning to translate these insights into novel immunotherapies. However, current technologies are often inadequate to promote further progress in our understanding of cellular and molecular immune pathways, and the available tools to 'engineer' immune cells to harness their full therapeutic potential are very limited. One major barrier is the lack of effective and robust intracellular delivery methods that could enable investigators to directly probe and manipulate intracellular pathways in primary immune cells. Modulating immune cell function through intracellular delivery of biomolecules has many potential applications. Delivery of macromolecules, such as polysaccharides, proteins, nucleic acids or multi-component nanostructures, to the cell cytoplasm can transiently or permanently alter cell function for research or therapeutic purposes. Herein, we propose to develop a vector-free microfluidic platform to deliver macromolecules and other payloads with high efficiency directly into the cytosol of primary mouse and human immune cells. The principle underlying this approach is temporary membrane disruption by rapid mechanical deformation, or squeezing, of immune cells to facilitate uptake of loading material in the fluid medium. Our preliminary data indicate that the proposed mechanical membrane disruption approach to intracellular delivery could potentially overcome the challenges encountered by traditional technologies, such as vector-based or electroporation-mediated systems, and enable delivery of a diversity of materials to manipulate cell function. Indeed, preliminary comparisons to alternative delivery methods demonstrate that our platform is capable of delivering functional protein and siRNA with equal or greater efficiency, while showing fewer off-target effects and maintaining genome integrity. We hypothesize that the proposed microfluidic cell squeezing platform can be harnessed to control immune cell fate and function. The key objectives of this project are to: i) generate an optimized library of microfluidic chips for use with a panel of relevant immune cell subsets, ii) characterize potential side-effects of the delivery system, and iii) use the novel capabilities of this technology to address the challenge of protein delivery. If successful, this work would facilitate the commercial launch of an enabling new technology platform and generate the preliminary data necessary to justify future work on platforms for ex vivo engineering of immune cells in therapeutic applications.

 描述（由申请人提供）：在过去的几十年中，免疫学家已经破译了哺乳动物免疫系统对抗或介导许多疾病的许多机制，临床医生开始将这些见解转化为新的免疫疗法。然而，目前的技术往往不足以促进我们对细胞和分子免疫途径的理解的进一步进展，并且“工程”免疫细胞以利用其全部治疗潜力的可用工具非常有限。一个主要的障碍是缺乏有效和稳健的细胞内递送方法，这些方法可以使研究人员直接探测和操纵原代免疫细胞中的细胞内途径。通过生物分子的细胞内递送来调节免疫细胞功能具有许多潜在的应用。将大分子如多糖、蛋白质、核酸或多组分纳米结构递送至细胞质可以暂时或永久地改变细胞功能以用于研究或治疗目的。在此，我们提出开发一种无载体的微流体平台，以高效地将大分子和其他有效载荷直接递送到原代小鼠和人免疫细胞的胞质溶胶中。该方法的基本原理是通过免疫细胞的快速机械变形或挤压来暂时破坏膜，以促进流体介质中装载材料的摄取。我们的初步数据表明，所提出的机械膜破裂细胞内递送方法可能会克服传统技术所遇到的挑战，例如基于载体或电穿孔介导的系统，并能够递送多种材料以操纵细胞功能。事实上，与替代递送方法的初步比较表明，我们的平台能够以相等或更高的效率递送功能性蛋白质和siRNA，同时表现出更少的脱靶效应并保持基因组完整性。 我们假设所提出的微流体细胞挤压平台可以用来控制免疫细胞的命运和功能。该项目的主要目标是：i）生成一个优化的微流控芯片库，用于一组相关的免疫细胞亚群，ii）表征递送系统的潜在副作用，iii）使用该技术的新功能来应对蛋白质递送的挑战。如果成功的话，这项工作将促进一个新技术平台的商业化推出，并产生必要的初步数据，以证明未来在治疗应用中对免疫细胞进行体外工程化平台的工作。