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Updated: Feb 28, 2026
Genome-Wide CRISPR Screen for Unveiling Radiosensitive and Radioresistant Genes
Published on: May 23, 2025
Rational Design of Immune Gene Therapy Combinations via In Vivo CRISPR Activation Screen of Tumor Microenvironment
Feifei Zhang1,2,3, Chuanpeng Dong1,2,3, Ryan D Chow1,2,3,4,5
1Department of Genetics, Yale University School of Medicine, New Haven, Connecticut.
Abstract:
The hostile tumor microenvironment (TME) remains a major challenge for cancer immunotherapy. In this study, we performed TME-targeted in vivo CRISPR activation (CRISPRa) screen to identify factors that promote antitumor immunity, culminating in rationally designed immune gene therapy combinations. Multiplexed activation of genes encoding antigen presentation, T-cell proliferation, costimulation, and migration (APCM) leads to enhanced antitumor responses. An APCM-focused CRISPRa screen in metastatic tumors identified Cd80, Tnfsf14, Cxcl10, Tnfsf18, Tnfsf9, and Ifng as top immunostimulatory candidates. Further optimization pinpointed Tnfsf9 (4-1BBL) + Ifng + Il12b (4II) as a potent therapeutic combination. Adeno-associated virus (AAV) 4II enhanced antigen presentation, T-cell activation, proliferation, cytotoxicity, and tumor infiltration. Preconditioning the TME with AAV-4II synergized with chimeric antigen receptor (CAR) and T-cell receptor (TCR) T-cell therapies to suppress primary and metastatic solid tumors in vivo. These findings establish TME-targeted CRISPRa screening as a rapid route to develop immune gene therapy combinations against solid tumors.
Significance:
By leveraging TME-focused in vivo CRISPRa screening, we identified immunomodulatory genes for rational AAV-based combinations that boost antitumor immunity. The optimized three-gene cocktail (4II) enhances antigen presentation and T-cell function and synergizes with adoptive T-cell therapies to improve immunotherapy efficacy in solid tumors and metastases.
Insights
This study used CRISPR activation screening to identify immune-boosting genes, leading to a novel combination therapy that enhances anti-tumor immunity and synergizes with cell therapies against solid tumors.
Area of Science:
- Immunology
- Oncology
- Gene Therapy
Background:
- The tumor microenvironment (TME) presents a significant barrier to effective cancer immunotherapy.
- Identifying factors that enhance anti-tumor immunity within the TME is crucial for developing novel therapeutic strategies.
Purpose of the Study:
- To conduct a TME-targeted in vivo CRISPR activation (CRISPRa) screen to discover genes promoting anti-tumor immunity.
- To develop rationally designed immune gene therapy combinations based on screening results.
- To evaluate the efficacy of a novel combination therapy in pre-clinical cancer models.
Main Methods:
- Performed a TME-targeted in vivo CRISPRa screen in metastatic tumors.
- Identified key immunostimulatory genes including CD80, TNFSF14, CXCL10, TNFSF18, TNFSF9, and IFNG.
- Optimized a therapeutic combination of TNFSF9 (4-1BBL), IFNG, and IL12B (AAV-4II).
- Assessed the effects of AAV-4II on antigen presentation, T cell functions, and tumor infiltration.
- Investigated the synergistic effects of AAV-4II with CAR-T and TCR-T cell therapies.
Main Results:
- Multiplexed activation of antigen presentation, T cell proliferation, co-stimulation, and migration (APCM) genes enhanced anti-tumor responses.
- The combination of TNFSF9 (4-1BBL) + IFNG + IL12B (4II) was identified as a potent therapeutic agent.
- AAV-4II significantly improved T cell activation, proliferation, cytotoxicity, and tumor infiltration.
- AAV-4II preconditioning synergized with CAR-T and TCR-T cell therapies to suppress solid tumors in vivo.
Conclusions:
- TME-targeted CRISPRa screening is an effective method for discovering immune gene therapy combinations.
- The developed AAV-4II therapy shows promise in enhancing anti-tumor immunity and potentiating cell-based cancer therapies.
- This approach offers a rapid route for developing novel immune gene therapies against solid tumors.
