{
  "abstract": "Background Chimeric antigen receptor (CAR) T cell therapies have achieved remarkable success in hematological malignancies, with seven products now approved. However, their broader clinical application is constrained by complex, costly, and patient-specific ex vivo manufacturing. Moreover, despite their success in blood cancers, CAR-T cell therapies have yet to demonstrate similar efficacy in solid tumors, largely due to the highly immunosuppressive tumor microenvironment (TME), which impairs immune cell trafficking, persistence, and function. To broaden the application of cell therapies and address the limitations of CAR-T cells, CAR-engineered macrophages and NK cells have emerged as alternative approaches, showing early signs of activity in solid tumors. Despite these advances, single-lineage ex vivo CAR approaches remain insufficient for most solid tumors. Engineering multiple cell types to fully harness the synergy between innate and adaptive immunity poses a promising strategy for solid tumor treatment.Methods We are developing an off-the-shelf, in vivo multi-lineage CAR platform using a helper-dependent adenovirus-based virus-like particle (VLP) system that selectively targets human CD46, a receptor highly expressed on hematopoietic stem and progenitor cells (HSPCs). Co-delivery with the Sleeping Beauty transposase enables stable integration of large transgenes up to 35 kb, facilitating sustained generation of engineered immune cells. To minimize off-target expression in progenitor or non-effector populations, we systematically screened and identified gene promoters that drive robust, lineage-specific CAR expression in mature effector immune cells. We ranked them based on strong activity in T and NK cells with minimal expression in B cells and HSPCs, and identified another promoter highly restricted to the myeloid lineage.Results CAR constructs under these promoters were validated in vitro using primary human and murine cells, generating functional CAR-T, CAR-NK, and CAR-macrophages with potent, antigen-dependent cytotoxicity. To assess expression in vivo, hCD46 transgenic mice were treated with VLPs encoding CARs under either ubiquitous (CAG) or lineage-restricted promoters. While the CAG promoter drove CAR expression across all immune cells, T/NK- and myeloid-restricted regulatory elements promoted CAR expression selectively in their respective lineages. Importantly, upon ex vivo analysis, these lineage-restricted CAR cells exhibited robust, target-dependent tumor cytotoxicity and cytokine production, comparable to CAG-driven CARs.Conclusions Our compelling data demonstrate that this platform has the potential to enable precise, scalable, and lineage-controlled in vivo CAR delivery. It addresses key limitations of current ex vivo and single-lineage CAR therapies, offering a promising, next-generation strategy for the effective treatment of solid tumors.Ethics Approval All animal studies were done under supervision and approval by IACUC",
  "authors": [
    {
      "affiliations": [
        "Ensoma, Boston, MA, USA"
      ],
      "name": "Alvin Pratama"
    },
    {
      "affiliations": [
        "Ensoma, Boston, MA, USA"
      ],
      "name": "Cristina Santoriello"
    },
    {
      "affiliations": [
        "Ensoma, Boston, MA, USA"
      ],
      "name": "Chirayu Chokshi"
    },
    {
      "affiliations": [
        "Ensoma, Boston, MA, USA"
      ],
      "name": "Yiwen Zhao"
    },
    {
      "affiliations": [
        "Ensoma, Boston, MA, USA"
      ],
      "name": "Ying-Cing Lin"
    },
    {
      "affiliations": [
        "Ensoma, Boston, MA, USA"
      ],
      "name": "Dhruv Varshney"
    },
    {
      "affiliations": [
        "Ensoma, Boston, MA, USA"
      ],
      "name": "Dennis Ramos Trinidad"
    },
    {
      "affiliations": [
        "Ensoma, Boston, MA, USA"
      ],
      "name": "Richard Davidson"
    },
    {
      "affiliations": [
        "Ensoma, Boston, MA, USA"
      ],
      "name": "Charles Fox"
    },
    {
      "affiliations": [
        "Ensoma, Boston, MA, USA"
      ],
      "name": "Victoria Blake"
    },
    {
      "affiliations": [
        "Ensoma, Boston, MA, USA"
      ],
      "name": "Alexey Seregin"
    },
    {
      "affiliations": [
        "Ensoma, Boston, MA, USA"
      ],
      "name": "Joe Salas"
    },
    {
      "affiliations": [
        "Ensoma, Boston, MA, USA"
      ],
      "name": "Robert Peters"
    },
    {
      "affiliations": [
        "Ensoma, Boston, MA, USA"
      ],
      "name": "Corinne Decker"
    },
    {
      "affiliations": [
        "SimnovaBio, Cambridge, MA, USA"
      ],
      "name": "Yinghua Wang"
    }
  ],
  "title": "1019 Discovery of lineage specific regulatory elements for development of in vivo CAR immune cell therapy via hematopoietic stem cell engineering",
  "uid": "0838b9d8-6bb4-52b3-a4be-1a32fa4dbb75"
}