AAV-SB-CRISPR Screening Identifies Genetic Checkpoints in Tumor-Infiltrating Primary Natural Killer Cells for CAR-NK Therapy

With the rapid advancement of biotechnology and gene editing technologies, increasing attention is being paid to enhancing the clinical potential of natural killer (NK) cells in cancer therapy. Despite their potential in clinical treatments, the success of NK cell therapies is restricted by multiple limitations. In a latest study published in Nature Biotechnology, researchers conducted an in vivo adeno-associated virus (AAV)-Sleeping Beauty (SB)-CRISPR screening based on primary NK cells, analyzing tumor-infiltrating NK (TINK) cells in four solid tumor mouse models. Concurrently, they performed single-cell transcriptome analysis on TINK cells, identifying previously unexplored NK cell subsets and differentially expressed TINK genes.

Researchers from Yale University, including Peng Lei, Paul A. Reinhart, Giacomo Sferluza, and Jiayang Luo, completed this study in 2030. They discovered that CALHM2 knockout (KO) NK cells exhibited enhanced cytotoxicity and tumor infiltration in both murine NK cells and human chimera antigen receptor (CAR)-NK cells. Transcriptome analysis revealed genes and pathways altered by CALHM2-KO. This study not only provides a deeper understanding of the biological functions of NK cells and cancer immunotherapy but also offers important insights into the application of NK cell-engineered immunotherapies.

Research Background

NK cells play a crucial role in tumor immunotherapy. Compared to traditional CAR-T cell therapy, CAR-NK therapy can utilize allogeneic NK sources without the risk of graft-versus-host disease (GVHD). However, existing NK cell-based immunotherapies face several challenges, including scarcity of cells, lower proliferation capacity, and reduced effectiveness, duration, or tumor infiltration.

Therefore, overcoming these limitations and improving the anti-tumor capacity of NK cells have become significant research topics. The purpose of this study was to address this issue by exploring the potential functions and clinical applications of NK cells at the genetic level using in vivo AAV-SB-CRISPR gene editing screening technology.

Research Process and Results

The research team designed a high-density single guide RNA (sgRNA) library for four tumor models and performed in vivo AAV-SB-CRISPR screening directly in primary NK cells. They extracted NK cells from tumors and spleens and collected transcriptome data. This approach identified intrinsic functional inhibitors of NK cells through CRISPR-based genetic modifications and revealed specific gene expression downregulation phenomena within TINK cells through single-cell transcriptome analysis.

Through these studies, researchers found that CALHM2 knockout significantly enhanced NK cells’ capability to kill cancer cells in vitro and their tumor infiltration ability in vivo. They also demonstrated that CALHM2 knockout could enhance tumor cell killing capacity in humanized CAR-NK92 cells. Moreover, pathway analysis showed that knockout of CALHM2 altered multiple pathways.

Research Conclusion and Significance

Peng Lei, Paul A. Reinhart, and their team successfully identified and validated CALHM2 as an intrinsic genetic checkpoint that restricts NK cell function through in vivo gene screening of NK cells. Knocking out CALHM2 significantly enhances NK cell functionality, bringing new insights into anti-cancer immunotherapies. This discovery provides new targets for NK cell engineering, and it holds significant scientific and clinical application value for the use of NK cells in cancer treatment.

Research Highlights

• Utilization of in vivo AAV-SB-CRISPR screening technology successfully identified the checkpoint gene CALHM2 that restricts NK cell functions.
• Single-cell transcriptome analysis revealed TINK cell subset differentiation.
• Verified that knocking out specific genes enhances NK and CAR-NK cell functions, increasing their anti-tumor potential.
• Brought a new perspective on the biological functions of NK cells and their potential in cancer treatment.

This study was completed by Peng Lei, Paul A. Reinhart, Giacomo Sferluza, Jiayang Luo, and colleagues from Yale University and was accepted for publication in Nature Biotechnology on May 10, 2024. Through comprehensive experimental design and innovative technology application, this study not only enriches the biological knowledge system of NK cells but also provides new strategies and insights for NK cell-based cancer therapies. It indicates that precisely regulating intrinsic inhibitory mechanisms in NK cells can effectively enhance their anti-cancer capabilities.