Rictor CRISPR Gene Editing by Lipid Nanoparticle Delivery Stimulates Anti-Tumor Immunity in Breast Cancer Liver Metastasis Model

Ali, Y.; Galbraith, T.; Abdelfattah, N. H.; Ziemys, A. H.; Faisal, M.; Wong, T.; Hashimoto, C.; Qian, X.; Cook, H.; Pandita, T.; Xu, Y.; Rosato, R.; Chen, S.-H.; Yun, K.; Leonard, F.

doi:10.1101/2025.06.26.661471

bioRxiv · 10.1101/2025.06.26.661471

Rictor CRISPR Gene Editing by Lipid Nanoparticle Delivery Stimulates Anti-Tumor Immunity in Breast Cancer Liver Metastasis Model

Ali, Y.·Galbraith, T.·Abdelfattah, N. H.·Ziemys, A. H.·Faisal, M.·Wong, T.·Hashimoto, C.·Qian, X.·Cook, H.·Pandita, T.·Xu, Y.·Rosato, R.·Chen, S.-H.·Yun, K.·Leonard, F.

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Abstract

Triple-negative breast cancer (TNBC) is a highly aggressive subtype, accounting for 10-15% of breast cancer cases in the United States. Liver metastases, common in advanced TNBC, are linked to especially poor outcomes, with a 5-year survival rate of just 11%. Although immune checkpoint inhibitors (ICIs) targeting PD-1 or PD-L1 show promise, durable responses in TNBC remain uncommon. This is largely due to a profoundly immunosuppressive tumor microenvironment (TME), driven by tumor-associated myeloid cells. Tumor-associated macrophages (TAMs) and neutrophils (TANs) polarize into immunosuppressive M2 and N2 phenotypes, respectively, suppressing T cell activity through cytokines, ROS, and checkpoint ligands such as VISTA. Myeloid-derived suppressor cells (MDSCs) further inhibit immunity by depleting nutrients and inducing regulatory T cells. As a result, despite its immunogenic features, TNBC remains resistant to immunotherapy due to persistent myeloid-mediated suppression. Here, we developed ionizable lipid nanoparticles (iLNPs) engineered to deliver the CRISPR-Cas12a ribonuclease complex targeting Rictor, a critical component of mTORC2, for in vivo reprograming of myeloid cells. The intravenous (IV) injection of CRISPR Rictor-targeting iLNP (CR-Ric-LNP) showed efficient uptake by circulating myeloid cells and accumulation into the breast cancer liver metastases. Notably, Rictor gene editing triggered pro-inflammatory activation of myeloid cells in the TME, enhancing antitumor responses. Single-cell RNA sequencing revealed that Rictor silencing treated samples showed induced rapid remodeling of the TME, with a significant reduction in immunosuppressive macrophages within 24 hours of treatment. Concurrently, cytotoxic T-cell populations exhibited increased interferon-gamma (Ifng) production, driving the emergence of specific myeloid clusters that were responsive to Interferon signaling, particularly in macrophages and neutrophils. A shift from an immunosuppressive to an inflammatory TME was further evidenced by an elevated Cxcl10/Spp1 ratio in myeloid cells. CR-Ric-LNP treatment also enhanced T-cell activation, reducing exhausted T cells and regulatory T cells (Tregs) while expanding natural killer (NK) cells, naive CD4+, and CD8+ T cells. These changes correlated with a decreased proportion of tumor cells and proliferating cells, ultimately leading to a significant survival benefit in a 4T1 breast cancer liver metastasis model. Our findings demonstrate that myeloid-targeted Rictor silencing reprograms the TME, promoting antitumor immunity and improving therapeutic outcomes. O_FIG O_LINKSMALLFIG WIDTH=199 HEIGHT=200 SRC="FIGDIR/small/661471v1_ufig1.gif" ALT="Figure 1"> View larger version (109K): org.highwire.dtl.DTLVardef@10a5e7aorg.highwire.dtl.DTLVardef@1c0c005org.highwire.dtl.DTLVardef@17ec6b5org.highwire.dtl.DTLVardef@27c507_HPS_FORMAT_FIGEXP M_FIG C_FIG

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Ali, Y., Galbraith, T., Abdelfattah, N. H., Ziemys, A. H., Faisal, M., Wong, T., Hashimoto, C., Qian, X., Cook, H., Pandita, T., Xu, Y., Rosato, R., Chen, S.-H., Yun, K., Leonard, F.. 2025-06-28. Rictor CRISPR Gene Editing by Lipid Nanoparticle Delivery Stimulates Anti-Tumor Immunity in Breast Cancer Liver Metastasis Model. https://doi.org/10.1101/2025.06.26.661471

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