College - Author 1
College of Engineering
Department - Author 1
Biomedical Engineering Department
Degree Name - Author 1
BS in Biomedical Engineering
Date
6-2025
Primary Advisor
Trevor Cardinal, College of Engineering, Biomedical Engineering Department
Additional Advisors
Emily Neal, College of Engineering/Bailey College of Science and Mathematics, Biomedical Engineering Department/Biological Sciences Department
Abstract/Summary
Cancer is a heterogeneous disease caused by unchecked cell proliferation. The disease contributed to 9.7 million deaths worldwide in 2022, with hematological malignancies (leukemias, lymphomas, and myelomas) accounting for over 1.4 million deaths in the United States from 1999 to 2020. This thesis focuses on the treatment of these cancers, with an emphasis on non-Hodgkin lymphomas (NHLs). R-CHOP (Rituximab-Cyclophosphamide, Doxorubicin hydrochloride, Oncovin/Vincristine, and Prednisone) is the standard of care first-line treatment for diffuse large B-cell lymphoma (DLBCL), the most common subtype of NHL, and encompasses a mixture of chemotherapy, immunotherapy, and corticosteroids. The R-CHOP regimen cures 50% to 70% patients, leaving 30% to 50% of patients with either relapsed or refractory (R/R) disease. For patients who are unresponsive to R-CHOP, high-dose (salvage) chemotherapy and autologous stem cell transplantation (HDC-ASCT) are typically the second-line treatment option. Patients who are resistant to these first- and second-line treatments are candidates for cellular immunotherapy, such as chimeric antigen receptor T cells (CAR T cells), as a third line (and sometimes second-line) treatment. A CAR consists of a single-chain variable fragment of the human IgG antibody, traditionally targeting CD19 or B-cell maturation antigen (BCMA), a costimulatory domain, and CD3 𝛇 chains, allowing a patient’s autologous T cells to recognize and kill cancer cells that have evaded the immune system. Despite the promise of these therapies, the lack of skilled personnel required to scale therapy development and the need for more precise CAR transgene insertion stand as obstacles in advancing this field. The Cell Therapy curriculum at Cal Poly aims to address these deficits by teaching students critical techniques relevant to cellular therapy development. The work in this thesis aims to improve upon a protocol for T cell electroporation to introduce students to a simple transfection model (electroporating GFP into Jurkat T cells) relevant to cellular therapy development. Electroporation can deliver the Cas9 plus gRNA system into cells for precise gene insertion, allowing this protocol to also lay the groundwork for future CRISPR transfection for stable gene expression. Because preliminary electroporation experiments yielded low cell viability (13% in a 10 μL electroporation reaction volume), I aimed to increase post-electroporation live cell percentages. I compared varying concentrations of the GFP plasmid payload, electroporation pulse parameters, and electroporation reaction volumes to determine the combination that yielded the highest cell viability with sufficient GFP expression. These experiments suggested that a GFP plasmid payload of 75 μg∙mL-1, a 100 μL electroporation reaction volume, and the initial recommended electroporation parameters from Thermo Fisher (1 pulse, 1700 V, and 20 ms pulse width) are the optimal conditions to increase post-electroporation live cell percentages over 3-fold from the preliminary experiment. Future work should investigate the longevity of GFP expression and explore transfecting a transgene and plasmids containing Cas9 plus gRNA to induce stable gene expression.
URL: https://digitalcommons.calpoly.edu/bmedsp/208