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Cellular and Molecular Bioengineering

Cellular and Molecular Bioengineering - Poster Session B

Poster H20 - Engineering a constitutive nuclear factor receptor 2 construct in chimeric antigen receptor T cells

Thursday, October 24, 2024

2:45 PM - 3:45 PM EST

Location: Exhibit Hall E, F & G

Presenting Author(s)

Jiayi Jiang (she/her/hers)

Student
University of Southern California
Los Angeles, California, United States

Co-Author(s)

CK

Cem Kilic, MS (he/him/his)

PhD Student
Univeristy of Southern California
Los Angeles, California, United States

Primary Investigator(s)

KS

Keyue Shen

Associate Professor
USC, California, United States

Introduction: T-cells engineered to express chimeric antigen receptors (CARs) successfully treated B-cell malignancies, but the hypoxic tumor microenvironment (TME) reduces effectiveness in solid tumors (1). Continuous stimulation under hypoxia drives T-cell exhaustion. Thus, strategies to alleviate TME-mediated CAR T-cell exhaustion can elicit a potent anti-cancer response against solid tumors. Transcription factors (TFs) regulate gene expression and cellular processes, playing significant roles in mediating metabolic and immunologic rewiring in the tumor microenvironment (TME). Targeting TFs to reprogram CAR-T cells enhances their metabolic fitness and antitumoral function. Nuclear Factor Receptor 2 (NRF2) is a transcription factor that regulates antioxidant enzymes. Overexpression of NRF2 reduces oxidative stress, improving T-cell function in the TME (2,3). In this study, we validated the feasibility of NRF2 expression in T-cells and optimized construct design and virus production protocols for creating NRF2 overexpressing CAR T-cells.

[1] Kotsafti A, Scarpa M, Castagliuolo I, Scarpa M. Reactive Oxygen Species and Antitumor Immunity-From Surveillance to Evasion. Cancers (Basel). 2020, 12(7):1748.
[2] Zimta, Alina-Andreea et al. “The Role of Nrf2 Activity in Cancer Development and Progression.” Cancers. 2019, 11(11): 1755.
[3] Ngo, V., & Duennwald, M. L. Nrf2 and Oxidative Stress: A General Overview of Mechanisms and Implications in Human Disease. Antioxidants (Basel, Switzerland). 2022, 11(12):2345.

Materials and

Methods: Plasmid Construction:
Different GD2 CAR constructs were cloned into the restriction enzymes digested FUGW backbone, utilizing a combination of PCR, gel electrophoresis, DNA isolation/purification, HiFi DNA assembly, and bacterial transformation to create different expression cassettes for virus production.

Lentivirus production and virus transduction:
After validating our construct with Sanger Sequencing, we utilized a second-generation virus production process to create lentiviral vectors to target the cells. We transfected LentiX cells with polyethylenimine (PEI), transfer plasmids, packing plasmid pspax2, and envelope plasmid pmd2.g. The viruses were concentrated in a LentiX concentrator and were collected for Jurkat or primary T-cell transduction.

Results, Conclusions, and Discussions: To validate the feasibility of NRF2 overexpression in T-cells, we created a NRF2 overexpression cassette FUGW-PGK-NRF2-2xP2A-mCherry, produced viruses and transduced Jurkat cells. Flow cytometry results confirmed the NRF2 expression in Jurkat T-cells (Fig. 1a), prompting the creation of CAR-NRF2 bicistronic vectors.
To optimize virus titers, we first attempted to optimize the size and expression levels of our CAR-NRF2 constructs by engineering GD2 CAR-28z-2xP2A-mCherry constructs with either the full length EF1-a promoter (L28z, 1200 bps) or the shorter Ef1-a core promoter (s28z, 200 bps), and used mCherry levels to quantify gene expression. We transduced primary T-cells and showed that L28z T-cells resulted in 4.7-fold higher mCherry MFI levels than s28z T-cells (Fig. 1b). The higher transduction efficiency of L28z favored the long EF1a promoter for virus production.
Transfection ratios of packaging, envelope, and transfer plasmids used during virus production significantly impacts virus titer. To optimize the virus production for constructs containing the long EF1a promoter (LEF1A), we transfected LentiX cells with 1:1:1 or 0.5:1:1 transfer:pspax2:pmd2.g ratios and tested the infectivity of the produced virus in Jurkat T-cells. Flow cytometry analysis showed a 46% and 22.9% mCherry+ Jurkat population for the 0.5:1:1 and 1:1:1, respectively (Fig. 1c).
In conclusion, we were able to validate NRF2 expression in Jurkat cells. Furthermore, we showed that the LEF1a promoter resulted in stronger gene expression relative to the sEF1a promoter. The virus production was optimized for GD2-CAR-2xP2A-mCherry constructs containing the LEF1a promoter. Next, we will create LEF1a-GD2 CAR-2xP2A-NRF2 construct, validate NRF2 expression using RT-PCR, and conduct functional consequences of NRF2 overexpression on GD2 CAR T-cell cytotoxicity and exhaustion under varying oxygenation levels.

Acknowledgements (Optional): This work has been supported by an NIH NCI grant R01CA220012, STOP CANCER Marni Levine Memorial Research Career Development Award, a USC Provost's undergraduate research fellowship, and USC Viterbi School of Engineering.