Evaluation of the Elements of Short Hairpin RNAs in Developing shRNA-Containing CAR T Cells.

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Publication Year:
2023
Authors:
Ryan Urak, Brenna Gittins, Citradewi Soemardy, Nicole Grepo, Lior Goldberg, Madeleine Maker, Galina Shevchenko, Alicia Davis, Shirley Li, Tristan Scott, Kevin V Morris, Stephen J Forman, Xiuli Wang
PubMed ID:
37345185
Funding Grants:
Public Summary:
Reprogramming the immune system to combat diseases, such as cancer, has slowly become a primary method of treatment. Of these methods, altering the immune cells known as T cells with chimeric proteins that redirect these cells to cancer cells has emerged to be the strongest methodology in fighting these diseases. Although this technology has led to immense benefits for these patient populations, the treatment isn’t perfect. The longevity of treatment could be avoided by cancer cells lacking the target. Because of this, we believe that combination treatment with weaker chemotherapy agents could be beneficial to patients. However, chemotherapy also will remove theses cells leading to poorer efficacy. We generated a platform that uses interfering RNA to knockdown the protein that processes this chemotherapy into its toxic form thus making the T cells resistant. We optimized this platform and showed it in not only in a cancer sense but also against HIV. We believe that this methodology will allow for CAR specific editing, which was incapable of happening at this current time.
Scientific Abstract:
Short hairpin RNAs (shRNAs) have emerged as a powerful tool for gene knockdown in various cellular systems, including chimeric antigen receptor (CAR) T cells. However, the elements of shRNAs that are crucial for their efficacy in developing shRNA-containing CAR T cells remain unclear. In this study, we evaluated the impact of different shRNA elements, including promoter strength, orientation, multiple shRNAs, self-targeting, and sense and antisense sequence composition on the knockdown efficiency of the target gene in CAR T cells. Our findings highlight the importance of considering multiple shRNAs and their orientation to achieve effective knockdown. Moreover, we demonstrate that using a strong promoter and avoiding self-targeting can enhance CAR T cell functionality. These results provide a framework for the rational design of CAR T cells with shRNA-mediated knockdown capabilities, which could improve the therapeutic efficacy of CAR T cell-based immunotherapy.