CAREER: The next generation intracellular delivery device for immunotherapy: The integration between ultrasonic transducer and microfluidic chip (UXuChip)

免疫疗法是一大类旨在刺激人体自身免疫系统以更好地识别和对抗癌症的疗法，它为癌症治疗开辟了一条令人兴奋的新途径。一类免疫疗法涉及改造T细胞，这是我们免疫系统中的关键细胞，以更好地识别癌细胞。由于每个患者对治疗都有独特的反应，因此可以使用一种称为过继细胞转移（ACT）疗法的个性化免疫疗法来提高成功率。ACT包括从患者自身的血液中提取T细胞，在体外（体外）进行工程化和大量培养，然后将工程化的T细胞重新注入患者体内。为了提供用于ACT治疗的工程T细胞，需要一种可靠的高通量技术将分子传递到细胞中，以控制基因和蛋白质的表达。这个CAREER项目的研究目标是克服现有传递技术的局限性，通过集成超声波换能器将分子传递到微流体系统中的细胞中。提出的细胞内递送装置，称为UXuChip，将提高分子递送效率，同时保持T细胞抗肿瘤活性。成功将有助于将免疫疗法从实验室应用到临床。该项目的教育计划的主要目标是通过扩大高中学生和更大的社区的教育以及从本科生到研究生研究人员的各级指导，将拟议的研究和教育活动结合起来。这项研究将与超声成像、微流控芯片开发和癌症治疗等令人兴奋的新教育机会的发展相结合。通过为期10周的研究实习，向高中生传授工程专业知识和知识，提高他们对工程和生物医学科学的兴趣。研究者的长期研究目标是利用工程原理开发癌症诊断和治疗的新技术和方法。为了实现这一目标，本CAREER项目的研究目标是开发下一代细胞内递送技术，以实现体外细胞为基础的治疗，从而彻底改变治疗策略。虽然许多免疫治疗计划显示出有希望的结果，但越来越多的治疗患者经历复发和复发，因为由于癌症异质性和表型多样性，每个患者对相同的药物有独特的反应。失败的原因是不需要的基因组和表型改变，工程T细胞不可控的细胞因子分泌，以及在细胞内递送后工程T细胞攻击正常细胞。为了解决这些不利的结果，该项目将利用基于T细胞工程的免疫疗法，提供一种新的细胞内递送技术，具有遗传、表型和功能稳定性，以及高细胞活力和递送效率。研究计划有三个目标。第一个目标是开发一种集成设备（UXuChip），该设备使用超声波换能器（UX）和微透镜复合体以及用于细胞操作的微流控芯片（uChip）。由单个铌酸锂（LNB）晶体产生的超声波（2 MHz至200 MHz）通过微透镜进行圆柱聚焦，将推动细胞垂直穿过收缩通道，破坏细胞膜，增加细胞内大分子传递的通透性。第二个目的是研究UXuChip在体外细胞水平上对基因和蛋白表达以及细胞因子分泌的影响，并将其与电穿孔进行比较。利用人/小鼠原代T细胞，优化电穿孔和UXuChip的参数，使细胞存活率和传递效率达到85%以上。与此同时，CRISPR-Cas9核糖核蛋白（RNP）将被开发用于消融PD-1， PD-1是t细胞表面限制抗肿瘤活性的受体/检查点蛋白。电穿孔和UXuChip处理后，在3小时和24小时后进行全基因组微阵列检测。研究结果将用于确定电穿孔和UXuChip治疗后错误表达的基因数量以及效果是否延长。研究将被设计来检验基因水平表达的变化可能影响细胞因子分泌的工作假设。第三个目标是通过使用UXuChip传递的RNPs和电穿孔来消融PD-1，并将工程T细胞注射到基因小鼠的肿瘤中，研究UXuChip工程T细胞的体内效应和治疗潜力。研究旨在验证这样一种假设，即消融后的PD-1不会与肿瘤细胞上的PD-L1结合，从而增加t细胞的抗肿瘤活性。UXuChip和电穿孔治疗T细胞的杀伤潜力将通过监测肿瘤肿块的大小进行比较。该奖项反映了美国国家科学基金会的法定使命，并通过使用基金会的知识价值和更广泛的影响审查标准进行评估，被认为值得支持。

Immunotherapy, a broad category of therapies designed to stimulate the body’s own immune system to better recognize and fight cancer, has opened an exciting new avenue for cancer therapy. One category of immunotherapy involves engineering T Cells, key cells in our immune systems, to better recognize cancer cells. As each patient has a unique response to treatment, and success can be improved using a personalized immunotherapy called adoptive cell transfer (ACT) therapy. ACT involves extraction of T Cells from a patient’s own blood, engineering and growing them in large numbers in vitro (outside the body), then reinfusing the engineered T Cells back into the patient. To supply engineered T Cells for ACT therapy, a reliable high-throughput technique is needed to deliver molecules into cells in order to control gene and protein expression. The research goal of this CAREER project is to overcome the limitations of existing delivery techniques by integrating ultrasonic transducers to deliver molecules into cells in a microfluidic system. The proposed intracellular delivery device, called the UXuChip, will improve the molecule delivery efficiency while maintaining T Cell antitumor activity. Success will help translate immunotherapy from bench to bedside. The primary goal of this project’s education initiative is to integrate the proposed research and educational activities by broadening the education of high school students and the greater community and mentoring at all levels from undergraduate students to post-graduate researchers. The research will be integrated with the development of exciting new educational opportunities on ultrasound imaging, microfluidic chip development, and cancer therapy. Engineering expertise and knowledge will be disseminated to high school students to promote their interest in engineering and biomedical sciences via 10-week research internships.The investigator's long-term research goal is to utilize engineering principles to develop novel technologies and approaches for cancer diagnosis and therapy. Towards this goal, the research aim of this CAREER project is to develop the next generation intracellular delivery technique to realize in vitro cell-based therapy to revolutionize therapeutic strategies. While many immunotherapy treatment plans have shown promising results, a growing number of treated patients experience recurrence and relapse because each patient has a unique response to the same drugs due to cancer heterogeneity and phenotypic diversity. Failure has been attributed to unwanted genomic and phenotypic alterations, uncontrollable cytokine secretion of engineered T cells, and attacking normal cells by engineered T cells in patients after the intracellular delivery. To address these unfavorable outcomes, the project will leverage T cell engineering-based immunotherapy by providing a novel intracellular delivery technique with genetic, phenotypic, and functional stability as well as high cell viability and delivery efficiency. The Research Plan is organized under three objectives. The FIRST Objective is development of an integrated device (UXuChip) using an ultrasonic transducer (UX) and microlens complex and a microfluidic chip (uChip) for cell manipulation. Ultrasound (2 MHz to 200 MHz) generated by a single lithium niobite (LNB) crystal and cylindrically focused by the microlens will push cells to pass through a constriction channel vertically to disrupt the cell plasma membrane to increase permeability for intracellular delivery of macromolecules. The SECOND Objective is investigation of in vitro cell-level influence of the UXuChip on gene and protein expressions and cytokine secretion, which will be compared with electroporation. Parameters of electroporation and UXuChip will be optimized to achieve cell viability and delivery efficiency over 85% using human / mouse primary T cells. At the same time, CRISPR-Cas9 ribonucleoprotein (RNP) will be developed to ablate PD-1, a receptor/checkpoint protein on the surface of T-Cells that limits antitumor activity. After treatment with electroporation and UXuChip, genome-wide microarray will be performed after 3 and 24 hours. Findings will be used to determine the number of genes that were misexpressed after electroporation and UXuChip treatment and if the effect is prolonged or not. Studies will be designed to test the working hypothesis that the changes in genetic level expression may affect cytokine secretion. The THIRD Objective is investigation of in vivo effects and assessing the therapeutic potential of engineered T cells by UXuChip by ablating PD-1 using RNPs delivered by UXuChip and electroporation and injecting the engineered T-cells into tumors created in congenic mice. Studies are designed to test the hypothesis that ablated PD-1 will not be conjugated with PD-L1 on tumor cells, resulting in increased T-cell antitumor activity. The killing potential of T cells treated by UXuChip and electroporation will be compared by monitoring the size of the tumor mass.This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.