Feifei Zhang et al. recently published a research article in Nature Biotechnology titled “Multiplexed inhibition of immunosuppressive genes with Cas13d for combinatorial cancer immunotherapy.” This paper describes a novel multiplexed, universal combinatorial immunotherapy approach using CRISPR-Cas13d to silence multiple endogenous immunosuppressive genes within the tumor microenvironment (TME), thereby promoting TME remodeling and enhancing antitumor immunity.

 

Background and Rationale

Cancer cells exploit the immunosuppressive TME to evade immune surveillance and resist therapeutic interventions. Immune checkpoint blockade (ICB) therapies, such as anti-PD-1/PD-L1 and anti-CTLA4 antibodies, have revolutionized cancer treatment by restoring T-cell-mediated immunity. However, the clinical success of ICB is limited, as many patients exhibit either intrinsic or acquired resistance, with single-agent therapies often insufficient to counteract the complexity of the TME. This limitation has driven the pursuit of combination therapies that can simultaneously target multiple immunoregulatory pathways. Conventional approaches to combinatorial immunotherapy face challenges, such as drug toxicity and regulatory complexity. The advent of CRISPR-Cas systems has introduced new possibilities for precise gene regulation. Cas13d, a smaller RNA-targeting member of the CRISPR family, enables simultaneous knockdown of multiple genes at the RNA level, offering an innovative approach to TME remodeling without introducing permanent changes to the genome. The rationale behind this study is grounded in the understanding that individual immunosuppressive genes, such as PD-L1, Galectin-9, Galectin-3, and CD47, play pivotal roles in maintaining the immunosuppressive nature of the TME. Targeting these genes in combination is hypothesized to generate synergistic antitumor effects by disrupting multiple layers of immune suppression. The authors proposed the Multiplex Universal Combinatorial Immunotherapy via Gene silencing (MUCIG) platform, which utilizes Cas13d to silence these genes at the RNA level.

 

Methods

Gene Selection and Pool Design:
From an initial list of 588 immunosuppressive genes identified via databases and literature, 19 targets were prioritized, with four—Cd274, Lgals9, Lgals3, and Cd47 (PGGC pool)—selected for their immune-modulating roles and consistent tumor expression.

CRISPR-Cas13d System Design:
Cas13d was chosen for its compact size, allowing a single AAV to deliver both the protein and gRNAs. Its RNA-targeting mechanism avoids DNA breaks, enhancing safety. Efficient gRNAs were computationally optimized and tested before finalizing.

AAV Vector Design:
An all-in-one AAV vector was engineered with U6 and EFS promoters and mutant direct repeats to enhance knockdown efficiency. AAV9 serotype was used for its ability to transduce tumor and immune cells.

Animal Models:
Syngeneic murine tumor models—E0771 (breast), Colon26 (colon), B16F10 (melanoma), Pan02 (pancreatic)—were used. AAV-Cas13d-PGGC was administered intratumorally, typically on days 5, 9, and 14.

Combination Therapy:
Combining AAV-Cas13d-PGGC with anti-GR1 antibodies enhanced efficacy by depleting MDSCs and neutrophils. Additional assays confirmed increased MHC-I expression, T-cell activation, and tumor cell susceptibility to T-cell-mediated killing.

 

Results and Key Data

1. Gene Silencing Efficiency:
Cas13d efficiently silenced the four target genes both at the RNA and protein levels, which displayed more significant efficiency compared to shRNA methods. Knockdown was specific, with minimal off-target effects, as confirmed by transcriptomic profiling. Enhanced knockdown of specific targets was observed with optimized direct repeats (Mut-DR). This method is effective across various syngeneic tumor models, showcasing broad efficacy in TME modulation.

2. Antitumor Efficacy:
AAV-Cas13d-PGGC significantly reduced tumor growth in multiple cancer models, including checkpoint-resistant E0771 breast cancer, Colon26 colon cancer, B16F10 melanoma, and immunologically “cold” Pan02 pancreatic cancer (Figure 1). Both B16F10 and Pan02 models are typically resistant to traditional ICB therapies, making these results particularly noteworthy. Tumor reduction correlated with improved immune cell infiltration and activity.

Figure 1. A four-gene combination immunotherapy AAV–Cas13d–PGGC demonstrates broad antitumor activity across diverse syngeneic cancer models.
Figure 1. A four-gene combination immunotherapy AAV–Cas13d–PGGC demonstrates broad antitumor activity across diverse syngeneic cancer models.

3. TME Remodeling:
AAV–Cas13d–PGGC therapy reshaped the immune landscape within tumors. Here are the observations:
Increased T cells: CD8+ and CD4+ T cells infiltrated tumors treated with AAV-Cas13d-PGGC.
Reduced suppressive populations: Profiling revealed downregulation of key immunosuppressive genes in the treated groups. Neutrophils and myeloid-derived suppressor cells (MDSCs) were depleted in treated tumors (Figure 2). Notably, specific pathways like neutrophil chemotaxis were consistently affected, aligning with the observed phenotypic changes.
Enhanced immune signaling: These changes correlated with enhanced production of IFN-γ by T cells, increased expression of MHC-I on tumor cells, and improved antigen presentation, indicating a shift toward a proinflammatory and antitumor TME.

  • Safety and Specificity:
    Treatment with a high-fidelity version of Cas13d (hfCas13d) maintained strong antitumor activity without causing systemic toxicity or weight loss. Minimal off-target effects were observed, confirmed through mRNA-seq analysis. No significant toxicity, such as weight loss or liver damage, was reported in treated models.
  • Combination Strategies:
    Combining AAV-Cas13d-PGGC with anti-GR1 antibodies to deplete both neutrophils and MDSCs enhanced tumor reduction compared to monotherapy.

 

4. Comparison with Alternative Methods

  • Versus Cas9:
    o Cas13d exhibited better transduction efficiency and efficacy without the genomic instability caused by DNA breaks from Cas9.
  • Versus shRNA:
    o Cas13d-mediated knockdown achieved stronger antitumor effects despite similar in vitro gene silencing efficiency.

 

5. Discovery of Synergistic Mechanisms

  • Immunogenicity:
    o Cas13d increased IFN-γ expression in CD8+ T cells, enhancing immune activation and antigen presentation.
    o MHC-I expression was upregulated, improving T-cell recognition and tumor cell killing.
  • Direct vs. Indirect Effects:
    oTumor growth reduction was largely immune-mediated, as shown by diminished efficacy in immune-deficient models.
Figure 2. AAV–Cas13d–PGGC treatment remodels the immunosuppressive TME.
Figure 2. AAV–Cas13d–PGGC treatment remodels the immunosuppressive TME.
Conclusion

The study establishes the MUCIG platform as a promising strategy for combinatorial cancer immunotherapy. By leveraging the RNA-targeting capabilities of Cas13d, the platform simultaneously disrupts multiple immunosuppressive pathways in the TME, leading to significant antitumor responses across diverse tumor models. The use of Cas13d, which avoids permanent DNA modifications, adds a layer of safety and reversibility, making it particularly attractive for clinical translation. The new discovery of the PGGC pool’s broad efficacy in checkpoint-resistant cancers represents a significant advance in immunotherapy. Moreover, traditional approaches, such as monoclonal antibodies and small molecules, often focus on single pathways, which can lead to incomplete responses or therapeutic resistance. The advent of RNA-targeting tools like Cas13d represents a paradigm shift, enabling precise and flexible modulation of multiple genes. This aligns with the growing interest in immunogenomics and personalized medicine, where therapies are tailored to the specific molecular and immunological profiles of individual tumors.

Reference:
1. Multiplexed inhibition of immunosuppressive genes with Cas13d for combinatorial cancer immunotherapy.
Zhang F, Chow RD, He E, Dong C, Xin S, Mirza D, Feng Y, Tian X, Verma N, Majety M, Zhang Y, Wang G, Chen S.
Nat Biotechnol. 2025 Jan 16.

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