{
  "abstract": "Background Conventional CAR T cell manufacturing relies predominantly on viral vectors, which have certain cost, production and payload capacity constraints. These limitations may hinder the integration of complex genetic circuits and delay clinical deployment. 1 2 To evaluate these challenges, we conducted studies on a non-viral gene delivery platform that could potentially yield same-day CAR T cell production without prior T cell activation, support large transgene payloads (>8 kb), and facilitate the incorporation of epigenetic regulatory circuits. This platform may streamline CAR T cell development processes while enhancing therapeutic versatility in complex tumor microenvironments.Methods We employed a transposon system in combination with electroporation to deliver CD19 CAR transgene cassettes. 3 4 CAR T cell functionality was assessed both in vitro and in vivo Nalm6 model compared to lentiviral vector (LVV)-engineered CAR T cells where applicable. Scalability was evaluated across input T cell numbers ranging from 5–10 × 106 to 50–100 × 106 cells.Results Non-viral-derived CD19 CAR T cells demonstrated comparable anti-tumor efficacy to their viral counterpart. We also observed increased anti-tumor activities in non-activated CAR T cells made by a shorter process compared to those made by the standard process. Using the platform for our studies, we achieved 40–60% transgene expression efficiency without the need for cell enrichment, suggesting a potentially improved delivery efficiency. Additionally, we observed scalability of the platform across a 10-fold range of input T cell numbers, supporting its potential for further development. We next evaluated the platform’s capacity to deliver large, multicistronic constructs, including a CAR payload of 8 kb and 10 kb and a context-dependent epigenetic regulator. The platform delivered these complex genetic payloads—resulting in 5–40% transgene expression. Notably, we observed that expression efficiency was dependent on sequence composition and design than on overall payload size. Functional validation confirmed IL-12 production upon CD19 CAR activation, demonstrating that the epigenetic circuit can operate within a single construct in primary T cells.Conclusions A non-viral electroporation-based platform may increase the payload, mitigate limitations of current viral systems, and could potentially enable rapid, scalable, and modular CAR T cell development processes. This approach may be further developed to expand the therapeutic scope of engineered T cells.References Tsai HC, Pietrobon V, Peng M, Wang S, Zhao L, Marincola FM, Cai Q. Current strategies employed in the manipulation of gene expression for clinical purposes. J Transl Med. 2022 Nov 18;20(1):535. doi: 10.1186/s12967-022-03747-3Ghassemi S, et al. Reducing Ex Vivo Culture Improves the Antileukemic Activity of Chimeric Antigen Receptor (CAR) T Cells. Cancer Immunol Res. 2018 Sep;6(9):1100-1109. doi: 10.1158/2326-6066.CIR-17-0405Ghassemi S, et al. Nat Biomed Eng. 2022 Feb;6(2):118-128. doi: 10.1038/s41551-021-00842-6Yang Z, et al. Nanoscale, antigen encounter-dependent, IL-12 delivery by CAR T cells plus PD-L1 blockade for cancer treatment. J Transl Med. 2023 Feb 28;21(1):158. doi: 10.1186/s12967-023-04014-9",
  "authors": [
    {
      "affiliations": [
        "Kite, a Gilead Company, Santa Monica, CA, USA"
      ],
      "name": "Hsing-Chuan Tsai"
    },
    {
      "affiliations": [
        "Kite, a Gilead Company, Santa Monica, CA, USA"
      ],
      "name": "Tiffany Jehng"
    },
    {
      "affiliations": [
        "Kite, a Gilead Company, Santa Monica, CA, USA"
      ],
      "name": "Graham Bilter"
    },
    {
      "affiliations": [
        "Kite, a Gilead Company, Santa Monica, CA, USA"
      ],
      "name": "Albert Millian"
    },
    {
      "affiliations": [
        "Kite, a Gilead Company, Santa Monica, CA, USA"
      ],
      "name": "Maoyu Peng"
    },
    {
      "affiliations": [
        "Kite, a Gilead Company, Santa Monica, CA, USA"
      ],
      "name": "Michelle So"
    },
    {
      "affiliations": [
        "Kite, a Gilead Company, Santa Monica, CA, USA"
      ],
      "name": "Violena Kaytazov"
    },
    {
      "affiliations": [
        "Kite, a Gilead Company, Santa Monica, CA, USA"
      ],
      "name": "Shashi Bala"
    },
    {
      "affiliations": [
        "Kite, a Gilead Company, Santa Monica, CA, USA"
      ],
      "name": "Alexander Falk"
    },
    {
      "affiliations": [
        "Kite, a Gilead Company, Santa Monica, CA, USA"
      ],
      "name": "Edward H Liao"
    },
    {
      "affiliations": [
        "Kite, a Gilead Company, Santa Monica, CA, USA"
      ],
      "name": "Tarinee Huang"
    },
    {
      "affiliations": [
        "Kite, a Gilead Company, Santa Monica, CA, USA"
      ],
      "name": "Nebi Wondyfraw"
    },
    {
      "affiliations": [
        "Kite, a Gilead Company, Santa Monica, CA, USA"
      ],
      "name": "Sophie Viaud"
    },
    {
      "affiliations": [
        "Kite, a Gilead Company, Santa Monica, CA, USA"
      ],
      "name": "Qi Cai"
    },
    {
      "affiliations": [
        "Kite, a Gilead Company, Santa Monica, CA, USA"
      ],
      "name": "David Barrett"
    }
  ],
  "title": "285 A non-viral engineering platform for rapid CAR T cell manufacturing and delivery of complex genetic payloads",
  "uid": "77dc0b0b-50aa-5e6f-b0fd-ed63967ed45c"
}