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

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

doi:10.3390/cancers15102848

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

Cancers (Basel). 2023 May 20;15(10):2848. doi: 10.3390/cancers15102848.

Authors

Affiliations

¹ Department of Hematology and Hematopoietic Cell Transplantation, City of Hope National Medical Center, Duarte, CA 91010, USA.
² Center for Gene Therapy, Beckman Research Institute, Duarte, CA 91010, USA.
³ Menzies Health Institute Queensland, School of Pharmacy and Medical Science, Griffith University, Gold Coast Campus, Southport, QLD 4215, Australia.

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.

Keywords: C-C chemokine receptor type 5 (CCR5); Patients Living With HIV (PLWH); Rev; Tat; chimeric antigen receptor (CAR); hypoxanthine phosphoribosyltransferase 1 (HPRT); shRNA-containing CARs; short hairpin RNA (shRNA).

Grants and funding

This work was supported by a grant from the California Institute for Regenerative Medicine (CIRM) (CLIN1-11223, PI: X.W) and CIRM scholar fellowship (PI: R.U). The Small Animal Imaging Core is supported by the National Cancer Institute of the National Institutes of Health (P30CA033572).