Genetic Engineering of Myeloid Cells to Treat Cancer - PROJECT SUMMARY/ABSTRACT Background: Despite the success of T cell therapies in treating hematologic malignancies, solid tumors remain a significant challenge due to their dense fibrotic stroma, immunosuppressive microenvironments, and antigen heterogeneity. Current adaptive cell therapy approaches struggle to overcome these barriers, highlighting the need for novel strategies. Myeloid cells, including macrophages and dendritic cells, possess inherent abilities to infiltrate tumors, secrete cytokines, and cross-present antigens within the tumor microenvironment, making them ideal candidates for therapeutic engineering. However, engineering these cells is technically challenging, and tumors often co-opt myeloid cells to suppress adaptive immunity. We aim to overcome these obstacles by applying advanced genetic engineering tools to reprogram myeloid cells, enhancing their tumoricidal activities and resistance to polarization into immunosuppressive states. Toolbox: We have developed a toolbox of new techniques that will allow us to overcome key engineering challenges in human myeloid cells. Recognizing that nucleofection can lead to significant toxicity and dysfunction, we have found that using enveloped delivery vehicles (EDVs) to deliver Cas9 preserves myeloid cell viability and functionality. Additionally, through a collaboration with the Landau lab, we have shown that lentivirus incorporating the Vpx system leads to highly efficient transduction of human myeloid cells. We have further engineered these lentiviruses to express a mutant VSVG and scFvs targeting myeloid-specific surface proteins, enabling selective transduction of specific myeloid compartments. Approach: 1) CRISPR Gene Editing: These tools will enable us to perform CRISPR-based knockout and base editing screens in primary human macrophages and dendritic cells to identify and modify key genes that regulate myeloid cell polarization and effector functions. In addition, we will explore base editing of genes with known roles and mutations in autoinflammatory diseases driven by overactive myeloid cells. 2) Synthetic Receptor (Innate-CAR) Library: To complement these gene editing approaches, we will design and test a library of synthetic receptors, which we call Innate-CARs, to enhance myeloid cell immunogenicity. These Innate-CARs will combine a tumor- specific scFv with intracellular domains from a variety of diverse innate immune receptors and intracellular proximal signaling molecules to optimize myeloid cell responses to tumor antigens. After we screen and identify top-performing Innate-CARs and gene edits, we will use a variety of immunodeficient and immunocompetent preclinical models to assess these enhancements in CAR-myeloid cell antitumor immunity. 3) In Vivo Engineering: Using our novel myeloid-targeting lentiviruses, we will evaluate the efficiency and efficacy of in vivo manufactured Innate-CAR myeloid cells in tumor-bearing humanized mouse models. Impact: By exploring the synthetic space of myeloid cell biology, we aim to push the boundaries of current immunotherapy paradigms and pave the way for new, effective living medicines to treat refractory solid tumors.
