Tong Yu

BioE PhD Defense Presentation

Date: Wednesday April 24, 2024

Time: 3:00 PM-5:00 PM

Location: EBB Krone - Children's Healthcare of Atlanta Seminar Room

Link: https://gatech.zoom.us/j/94388191557?pwd=VUxqUmI2YkFsMURRSHEwcC85dW1VQT09

Committee Members:

Todd Sulchek, PhD (Advisor)

Sunil Raikar, MD

Gabe Kwong, PhD

James Dahlman, PhD

Wilbur Lam, PhD

A Biomechanics-Based Delivery Strategy To Primary Immune Cells For Generating Cell Therapy With Multiple Gene Knockout

 Abstract: Adaptive T cell therapy has emerged as a promising strategy in cancer treatment, utilizing synthetic receptor modified T cells to specially target tumor antigens. Despite successes, challenges persist, including the need for multiplexed gene editing in production of allogeneic T cell product, expanding application to T cell malignancies, and overcoming T cell dysfunction. These challenges require new technologies that lead to safer and efficient multiplexed gene editing techniques to lead to improved therapies. Currently, multiplexed gene editing is performed in one process step, raising concerns regarding chromosome translocations. This thesis addresses safer and more efficient multiplexed gene editing by leveraging the innovative microfluidic volume exchange for cell transfection (VECT) platform. To achieve efficient and reproducible delivery of gene editing cargo to primary T cells, we propose to understand device and intrinsic cellular attributes that significantly impact delivery outcome. Then, we design optimal devices for sequential gene editing of primary T cells in CAR (Chimeric Antigen Receptor) T engineering pipeline, focusing on the reduction of chromosomal translocation. We hypothesize sequential multiplexed gene editing results in lower chromosomal translocation and improved T cell persistence. This study addresses the goals through 3 aims. Aim 1 focuses on identifying critical design elements (CDEs) for VECT devices, revealing device design and operational factors influencing delivery to primary T cells. Aim 2 demonstrates VECT's capability in functional Cas9 delivery and sequential gene editing of CAR T cells. Aim 3 focuses on intrinsic cell mechanics to reveal cell biomechanics' contributions to delivery efficiency. In completing the study, we created two easy fabrication methods to reproducibly generate high delivery to T cells, Then, we demonstrated an application of VECT to deliver CRISPR/Cas9 to mediate gene editing in T cells. VECT was shown to be capable of generating highly efficient and viable TCR and B2M knockout T cells in both batch and sequential workflow. Importantly, VECT sequential editing is shown to reduce the frequency of chromosomal translocations. Interestingly, we identified a combined effect of strain rate and acceleration to significantly improve delivery; and identified cell stiffness as an intrinsic determinant of delivery efficiency. Overall, this study underscores VECT's potential in industrial-scale multiplexed gene editing of T cells with improved safety profile.