Rational Design of Immune Gene Therapy Combinations via In Vivo CRISPR Activation Screen of Tumor Microenvironment...

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.