{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,9,5]],"date-time":"2026-09-05T11:28:46Z","timestamp":1788607726374,"version":"build-2803163510"},"reference-count":52,"publisher":"BMJ","issue":"5","license":[{"start":{"date-parts":[[2026,5,13]],"date-time":"2026-05-13T00:00:00Z","timestamp":1778630400000},"content-version":"unspecified","delay-in-days":12,"URL":"https:\/\/creativecommons.org\/licenses\/by-nc\/4.0\/"}],"funder":[{"DOI":"10.13039\/100019470","name":"Lazio Innova","doi-asserted-by":"crossref","award":["F89J23001120007"],"award-info":[{"award-number":["F89J23001120007"]}],"id":[{"id":"10.13039\/100019470","id-type":"DOI","asserted-by":"crossref"}]},{"name":"AIRC","award":["IG-27070"],"award-info":[{"award-number":["IG-27070"]}]}],"content-domain":{"domain":["bmj.com"],"crossmark-restriction":true},"short-container-title":["J Immunother Cancer"],"accepted":{"date-parts":[[2026,4,22]]},"published-print":{"date-parts":[[2026,5]]},"abstract":"<jats:sec>\n                    <jats:title>Background<\/jats:title>\n                    <jats:p>Immune checkpoint inhibitors have transformed cancer therapy, yet many patients fail to respond, underscoring the need for complementary strategies. Personalized neoantigen cancer vaccines (NCVs), which stimulate highly specific T-cell responses against tumor-specific mutations, are a promising approach now advancing in clinical development.<\/jats:p>\n                  <\/jats:sec>\n                  <jats:sec>\n                    <jats:title>Methods<\/jats:title>\n                    <jats:p>We developed a clinically scalable platform based on high-purity linear hairpin DNA (hpDNA), produced by an entirely cell-free enzymatic method and delivered intramuscularly by electroporation (EP). An M20-hpDNA vaccine, encoding 20 neoantigens from the MC38 tumor model, was benchmarked against a plasmid DNA (pDNA) counterpart and a pseudouridine-modified messenger RNA (mRNA) version delivered by EP. Immune responses were characterized by enzyme-linked immunospot, flow cytometry, bulk RNA sequencing, and single-cell RNA\/T-cell receptor sequencing. Antitumor activity was assessed in prophylactic, therapeutic, and metastatic mouse models.<\/jats:p>\n                  <\/jats:sec>\n                  <jats:sec>\n                    <jats:title>Results<\/jats:title>\n                    <jats:p>\n                      hpDNA and pDNA vaccines elicited robust and comparable CD8\n                      <jats:sup>+<\/jats:sup>\n                      and CD4\n                      <jats:sup>+<\/jats:sup>\n                      neoantigen-specific T-cell responses, leading to dose-dependent protection in both prophylactic and therapeutic settings. In contrast, pseudouridine-modified mRNA vaccine delivered by EP failed to elicit detectable immunity despite measurable transgene expression. Transcriptomic profiling revealed that hpDNA, but not EP-delivered mRNA, triggered a pro-inflammatory signature, including Il6, Ccl4, Cxcl2, Cd80, and Spp1. Single-cell sequencing of Adpgk-specific CD8\n                      <jats:sup>+<\/jats:sup>\n                      T cells showed expansion of memory subsets and unique hyperexpanded clonotypes. Functionally, M20-hpDNA prevented lung metastases in a mixed-modality protocol and synergized with \u03b1-cytotoxic T-lymphocyte associated protein 4 therapy. Efficacy was further confirmed with C20-hpDNA, encoding 20 CT26-specific neoantigens.\n                    <\/jats:p>\n                  <\/jats:sec>\n                  <jats:sec>\n                    <jats:title>Conclusions<\/jats:title>\n                    <jats:p>These findings demonstrate that hpDNA delivered by EP is a versatile, potent, and clinically scalable platform for personalized NCVs. Under the delivery conditions tested here, EP-delivered, non-formulated mRNA did not elicit detectable antigen-specific T-cell responses, whereas hpDNA induces productive inflammation and durable T-cell memory. These results highlight the importance of delivery context in shaping vaccine immunogenicity and support further development of hpDNA as a safe and competitive strategy for individualized cancer immunotherapy. Importantly, our data do not challenge the well-established immunogenicity and clinical activity of formulated mRNA vaccines (eg, mRNA-lipid nanoparticle) in infectious disease and cancer settings.<\/jats:p>\n                  <\/jats:sec>","DOI":"10.1136\/jitc-2025-013812","type":"journal-article","created":{"date-parts":[[2026,5,13]],"date-time":"2026-05-13T14:30:22Z","timestamp":1778682622000},"page":"e013812","update-policy":"https:\/\/doi.org\/10.1136\/crossmarkpolicy","source":"Crossref","is-referenced-by-count":3,"title":["Synthetic DNA vaccine platform elicits potent immunity where electroporated naked-mRNA is non-immunogenic"],"prefix":"10.1136","volume":"14","author":[{"ORCID":"https:\/\/orcid.org\/0000-0002-9979-1142","authenticated-orcid":false,"given":"Claudia","family":"Tonini","sequence":"first","affiliation":[{"name":"Neomatrix, Rome, 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