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Multiplexed inhibition of immunosuppressive genes with Cas13d for combinatorial cancer immunotherapy

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Abstract

The complex nature of the immunosuppressive tumor microenvironment (TME) requires multi-agent combinations for optimal immunotherapy. Here we describe multiplex universal combinatorial immunotherapy via gene silencing (MUCIG), which uses CRISPR–Cas13d to silence multiple endogenous immunosuppressive genes in the TME, promoting TME remodeling and enhancing antitumor immunity. MUCIG vectors targeting four genes delivered by adeno-associated virus (AAV) (Cd274/Pdl1, Lgals9/Galectin9, Lgals3/Galectin3 and Cd47; AAV-Cas13d-PGGC) demonstrate significant antitumor efficacy across multiple syngeneic tumor models, remodeling the TME by increasing CD8+ T-cell infiltration while reducing neutrophils. Whole transcriptome profiling validates the on-target knockdown of the four target genes and shows limited potential off-target or downstream gene alterations. AAV-Cas13d-PGGC outperforms corresponding shRNA treatments and individual gene knockdown. We further optimize MUCIG by employing high-fidelity Cas13d (hfCas13d), which similarly showed potent gene silencing and in vivo antitumor efficacy, without weight loss or liver toxicity. MUCIG represents a universal method to silence multiple immune genes in vivo in a programmable manner, offering broad efficacy across multiple tumor types.

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Fig. 1: Multiplexed Cas13d repression of immunosuppressive genes as combinatorial cancer immunotherapy.
Fig. 2: A four-gene combination immunotherapy AAV–Cas13d–PGGC demonstrates broad antitumor activity across diverse syngeneic cancer models.
Fig. 3: AAV–Cas13d–PGGC treatment remodels the immunosuppressive TME.
Fig. 4: Cas13d-mediated repression of immunosuppressive genes demonstrates superior antitumor efficacy compared with shRNAs.
Fig. 5: AAV–Cas13d is immunogenic in vivo.
Fig. 6: Optimized combinatorial targeting of immunosuppressive genes by hfCas13d promotes antitumor immunity.

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Data availability

All data generated or analyzed during this study are included in this Article and its Supplementary Information. Data and statistics of nonNGS experiments are provided in an Excel file in the Supplementary Information. Raw sequencing data of single-cell RNA-Seq, pool screen and off-target bulk RNA-Seq have been deposited to the Gene Expression Omnibus with accession number GSE269516 ref. 62. Processed data for genomic sequencing and gene expression (NGS experiments) are provided as processed quantifications in Supplementary Data. All other data and materials that support the findings of this research are available to the academic community upon reasonable request to the corresponding author. Source data are provided with this paper.

Code availability

All codes used for this study are available via Code Ocean at https://codeocean.com/capsule/0336604/tree/v1 (ref. 63).

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Acknowledgements

We thank all members of the Chen laboratory as well as various colleagues in Yale Genetics, SBI, CSBC, MCGD, Immunobiology, BBS, YCC, YSCC and CBDS for assistance and/or discussions. We thank various Yale Core Facilities such as YARC, WCFC, YCGA, HPC and WCAC for technical support. S.C. is supported by NIH/NCI/NIDA (grant numbers DP2CA238295, R01CA231112, R33CA225498, RF1DA048811 and R33CA281702), DoD (grant numbers W81XWH-17-1-0235, W81XWH-20-1-0072, W81XWH-21-1-0514 and HT94252310472), a Damon Runyon Dale Frey Award (DFS-13-15), the Melanoma Research Alliance (grant numbers 412806 and 16-003524), the Cancer Research Institute (Cancer Research Institute Lloyd J. Old STAR Award (CRI4964), CLIP), the AACR (17-20-01-CHEN), The V Foundation (grant number V2017-022), the Alliance for Cancer Gene Therapy, Sontag Foundation (DSA), the Pershing Square Sohn Cancer Research Alliance, Dexter Lu, the Ludwig Family Foundation, the Blavatnik Family Foundation and the Chenevert Family Foundation. G.W. is supported by CRI Irvington and RJ Anderson postdoctoral fellowships. R.C. is supported by NIH MSTP training grant number T32GM007205 and NRSA fellowship (grant number F30CA250249). C.D. is supported by Boehringer Ingelheim Biomedical Data Science Fellowship. N.V. is supported by American Board of Radiology’s B. Leonard Holman Research Pathway Fellowship and the ASTRO Radiation Oncology Seed Grant.

Author information

Author notes

  1. Guangchuan Wang

    Present address: Key Laboratory of Multi-Cell Systems, Shanghai Institute of Biochemistry and Cell Biology, Center for Excellence in Molecular Cell Science, Chinese Academy of Sciences, Shanghai, China

  2. These authors contributed equally: Feifei Zhang, Ryan D. Chow.

  3. These authors jointly supervised this work: Guangchuan Wang, and Sidi Chen.

Authors and Affiliations

  1. Department of Genetics, Yale University School of Medicine, New Haven, CT, USA

    Feifei Zhang, Ryan D. Chow, Emily He, Chuanpeng Dong, Shan Xin, Daniyal Mirza, Yanzhi Feng, Xiaolong Tian, Nipun Verma, Medha Majety, Yueqi Zhang, Guangchuan Wang & Sidi Chen

  2. System Biology Institute, Yale University, West Haven, CT, USA

    Feifei Zhang, Ryan D. Chow, Emily He, Chuanpeng Dong, Shan Xin, Daniyal Mirza, Yanzhi Feng, Xiaolong Tian, Nipun Verma, Medha Majety, Yueqi Zhang, Guangchuan Wang & Sidi Chen

  3. Center for Cancer Systems Biology, Yale University, West Haven, CT, USA

    Feifei Zhang, Ryan D. Chow, Emily He, Chuanpeng Dong, Shan Xin, Daniyal Mirza, Yanzhi Feng, Xiaolong Tian, Nipun Verma, Medha Majety, Yueqi Zhang, Guangchuan Wang & Sidi Chen

  4. M.D.–Ph.D. Program, Yale University, West Haven, CT, USA

    Ryan D. Chow & Sidi Chen

  5. Molecular Cell Biology, Genetics, and Development Program, Yale University, New Haven, CT, USA

    Ryan D. Chow & Sidi Chen

  6. Yale College, New Haven, CT, USA

    Emily He, Daniyal Mirza, Medha Majety & Sidi Chen

  7. Immunobiology Program, Yale University, New Haven, CT, USA

    Yanzhi Feng & Sidi Chen

  8. Department of Therapeutic Radiology, Yale University School of Medicine, New Haven, CT, USA

    Nipun Verma

  9. Department of Neurosurgery, Yale University School of Medicine, New Haven, CT, USA

    Sidi Chen

  10. Yale Comprehensive Cancer Center, Yale University School of Medicine, New Haven, CT, USA

    Sidi Chen

  11. Yale Stem Cell Center, Yale University School of Medicine, New Haven, CT, USA

    Sidi Chen

  12. Yale Center for Biomedical Data Science, Yale University School of Medicine, New Haven, CT, USA

    Sidi Chen

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Contributions

Conceptualization: S.C. Design: F.Z., G.W., R.C. and S.C. Experimental lead: F.Z., G.W., and E.H. Analytic lead: R.C. and C.D. Experimental and analytic assistance and support: S.X., D.M., Y.F., M.M., X.T. and Y.Z. Paper preperation: F.Z., R.C., G.W., C.D., S.X., N.V. and S.C. Supervision and funding: S.C.

Corresponding authors

Correspondence to Guangchuan Wang or Sidi Chen.

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Competing interests

S.C. is a (co)founder of EvolveImmune Tx, Cellinfinity Bio, MagicTime Med and Chen Consulting. A patent application (WO2023196711A3, worldwide) has been filed by Yale University related to this study. The other authors declare no competing interests.

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Source data and statistics of nonNGS-type data in the Sup_excel_files.zip file.

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Zhang, F., Chow, R.D., He, E. et al. Multiplexed inhibition of immunosuppressive genes with Cas13d for combinatorial cancer immunotherapy. Nat Biotechnol (2025). https://doi.org/10.1038/s41587-024-02535-2

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