Simultaneous engineering of natural killer cells for CAR transgenesis and CRISPR-Cas9 knockout using retroviral
Dong-Hyeon Jo1,2, Shelby Kaczmarek1,2, Oksu Shin1
1Department of Biochemistry, Microbiology, and Immunology, Faculty of Medicine, University of Ottawa, Ottawa, ON, Canada.
Abstract:
Natural killer (NK) cells are potent cytotoxic innate lymphocytes that can be used for cancer immunotherapy. Since the balance of signals from activating and inhibitory receptors determines the activity of NK cells, their anti-tumor activity can be potentiated by overexpressing activating receptors or knocking out inhibitory receptors via genome engineering, such as chimeric antigen receptor (CAR) transgenesis and CRISPR-Cas9-mediated gene editing, respectively. Here, we report the development of a one-step strategy for CRISPR-Cas9-mediated gene knockout and CAR transgenesis in NK cells using retroviral particles. We generated NK cells expressing anti-epidermal growth factor receptor (EGFR)-CAR with simultaneous TIGIT gene knockout using single transduction and evaluated the consequence of the genetic modifications in vitro and in vivo. Taken together, our results demonstrate that retroviral particle-mediated engineering provides a strategy readily applicable to simultaneous genetic modifications of NK cells for efficient immunotherapy.
Insights
This study presents a novel one-step method for engineering natural killer (NK) cells for cancer immunotherapy. The technique simultaneously introduces chimeric antigen receptors (CARs) and knocks out inhibitory genes, enhancing anti-tumor activity.
Area of Science:
- Immunology
- Cancer Research
- Genetic Engineering
Background:
- Natural killer (NK) cells are crucial cytotoxic lymphocytes for innate immunity and cancer immunotherapy.
- NK cell activity is regulated by a balance of activating and inhibitory receptor signals.
- Enhancing NK cell anti-tumor activity requires genetic modification strategies.
Purpose of the Study:
- To develop a one-step strategy for simultaneous gene knockout and CAR transgenesis in NK cells.
- To engineer NK cells with enhanced anti-tumor potential for immunotherapy.
Main Methods:
- Utilized retroviral particles for a one-step CRISPR-Cas9-mediated gene knockout and CAR transgenesis.
- Generated NK cells expressing anti-epidermal growth factor receptor (EGFR)-CAR with simultaneous TIGIT gene knockout.
- Evaluated the efficacy of genetically modified NK cells in vitro and in vivo.
Main Results:
- Successfully developed a one-step retroviral particle-mediated strategy for multiplex genetic modification of NK cells.
- Demonstrated the generation of NK cells with simultaneous CAR expression and TIGIT knockout.
- Confirmed the functional consequences of these genetic modifications in vitro and in vivo.
Conclusions:
- Retroviral particle-mediated engineering offers a streamlined approach for simultaneous genetic modifications in NK cells.
- This strategy is readily applicable for developing potent NK cell-based immunotherapies.
- The developed method enhances NK cell anti-tumor activity for improved cancer treatment.
