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June 28th, 2025
Version: 1
Houston Methodist Research Institute
bioengineering
biorxiv

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

Ali, Y.Open in Google Scholar•Galbraith, T.Open in Google Scholar•Abdelfattah, N. H.Open in Google Scholar•Ziemys, A. H.Open in Google Scholar•Faisal, M.Open in Google Scholar•Wong, T.Open in Google Scholar•Hashimoto, C.Open in Google Scholar•Qian, X.Open in Google Scholar•Cook, H.Open in Google Scholar•Pandita, T.Open in Google Scholaret al.

Targeting myeloid cells to remodel the immunosuppressive tumor microenvironment (TME) represents a promising strategy for cancer therapy. Here, we developed ionizable lipid nanoparticles (LNPs) engineered to deliver the CRISPR-Cas12a ribonuclease complex targeting Rictor, a critical component of mTORC2 and a key immunosuppression factor, for in vivo reprogramming of myeloid cells. Systemic delivery of CRISPR Rictor-targeting LNPs (CR-Ric-LNP) enabled efficient uptake by circulating myeloid cells, which were then recruited into breast cancer liver metastases. Notably, Rictor gene editing triggered pro-inflammatory activation in macrophages, enhancing their antitumor responses. Single-cell RNA sequencing revealed that Rictor silencing induced rapid remodeling of the TME, with 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 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 to Spp1 ratio in myeloid cells. CR-Ric-LNP treatment also enhanced T-cell activation, reducing exhausted T cells and regulatory T cells, 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.

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