{
  "abstract": "Background Personalized neoantigen immunotherapies show strong promise in early clinical trials, with mRNA-based platforms emerging as versatile, safe, and cost-effective. These therapies require rapid, small-batch mRNA production within 6–8 weeks from biopsy to administration. A key bottleneck is the supply of high-quality DNA templates for in vitro transcription (IVT). Currently, plasmid DNA (pDNA), produced via bacterial fermentation in large-scale bioreactors, faces 9–12 month lead times with inflexible batch sizes, far exceeding the few milligrams required per patient. This study introduces a cell-free DNA platform suitable for rapid, scale-out manufacture and evaluates its integration with a modular, functionalized lipid nanoparticle (LNP) delivery system.Methods Synthetic opDNA® is a linear, double-stranded DNA construct, open at the 3’ end, enabling direct use in IVT reactions without enzymatic linearization. Using high-fidelity enzymatic amplification (error rate: 3.75×10 – 9), a 120 bp polyA tail was directly encoded, removing the need for enzymatic tailing. opDNA® or pDNA encoding 20 neoantigens against the MC38 murine tumour model were transcribed and compared for mRNA quality and in vivo immunogenicity.For delivery, mRNA was encapsulated in Hermes® LNPs that combine a cationic scaffold with conventional lipid components. This modular design allows the inclusion of functional modalities, e.g. targeting ligands, in a one-step microfluidics method without additional conjugation steps. Here, the cationic scaffold was used to incorporate an adjuvant modality into the formulation.Results Capillary electrophoresis confirmed that opDNA® templates produced a homogenous polyA tail population and, that opDNA®-derived mRNA showed higher integrity (88.1%) than pDNA-derived (82.7%). Immunogenicity and tumour protection of Hermes® nanoparticles encapsulating the opDNA®-derived mRNA was benchmarked against a clinically validated LNP formulation in a therapeutic prime-boost regimen combined with αPDL1. Both formulations induced comparable T cell responses and tumour growth inhibition.Functionalization of Hermes® nanoparticles significantly enhanced immunogenicity and efficacy. Intramuscular vaccination elicited increased T cell responses versus non-functionalized formulations. Upon tumour challenge, 60% of animals receiving the functionalized Hermes® remained tumour-free, compared to 0% in the scaffold-only group.Conclusions opDNA® enables rapid, high-quality DNA template production suited to the demands of personalized mRNA immunotherapy. Combined with Hermes® LNPs, this approach offers a fast, flexible manufacturing platform that drives potent immune activation. Together, these innovations reduce the time and cost associated with therapeutic development and open the door to more agile, patient-centric treatments in oncology. The future of cancer care is increasingly personalized and novel manufacturing approaches must be adopted now to facilitate their journey to clinic.Ethics Approval All animal experiments were conducted in Italy in accordance with the European Directive 2010/63/EU on the protection of animals used for scientific purposes. All procedures were designed to minimize animal suffering and the number of animals used, in compliance with the principles of the 3Rs (Replacement, Reduction, and Refinement).",
  "authors": [
    {
      "affiliations": [
        "4Basebio, Cambridge, Cambridgeshire, UK"
      ],
      "name": "Emily R Young"
    },
    {
      "affiliations": [
        "Neomatrix, Roma, Italy"
      ],
      "name": "Ilaria Esposito"
    },
    {
      "affiliations": [
        "4Basebio, Cambridge, Cambridgeshire, UK"
      ],
      "name": "Ashish Dhir"
    },
    {
      "affiliations": [
        "Neomatrix, Roma, Italy"
      ],
      "name": "Claudia Tonini"
    },
    {
      "affiliations": [
        "Neomatrix, Roma, Italy"
      ],
      "name": "Mauro Esposito"
    },
    {
      "affiliations": [
        "Neomatrix, Roma, Italy"
      ],
      "name": "Andrea Bianchi"
    },
    {
      "affiliations": [
        "4Basebio, Cambridge, Cambridgeshire, UK"
      ],
      "name": "Zoe Whiffen"
    },
    {
      "affiliations": [
        "Neomatrix, Roma, Italy"
      ],
      "name": "Fabio Palombo"
    },
    {
      "affiliations": [
        "4Basebio, Cambridge, Cambridgeshire, UK"
      ],
      "name": "Amy Walker"
    }
  ],
  "title": "1204 Synthetic linear DNA and functionalized LNPs enable rapid manufacture and delivery of personalized mRNA immunotherapies",
  "uid": "cd28df60-e499-53a2-a043-c60f510193b7"
}