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.
