{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,7,3]],"date-time":"2026-07-03T05:18:48Z","timestamp":1783055928565,"version":"3.54.6"},"reference-count":31,"publisher":"MDPI AG","issue":"7","license":[{"start":{"date-parts":[[2026,6,30]],"date-time":"2026-06-30T00:00:00Z","timestamp":1782777600000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["Pharmaceutics"],"abstract":"<jats:p>Background: Scalability and cost remain major manufacturing barriers limiting broad patient access to adeno-associated virus (AAV) gene therapies. While capsid engineering has advanced vector biology, comparatively fewer innovations have addressed fundamental upstream productivity constraints. Transient triple-plasmid transfection is still the dominant AAV production platform and relies on large bacterial backbone plasmids that impose DNA burden and contribute significantly to the cost-of-goods. Methods: In this study, we evaluated a compact NanoplasmidTM DNA system (Aldevron) as a structural redesign of the transfection substrate to enhance upstream productivity. Conventional pUC-based triple-plasmid systems were compared to fully substituted NanoplasmidTM equivalents across suspension HEK293 production platforms optimised via response surface Design of Experiments. Hybrid plasmid configurations were also constructed to assess component-level contributions. Results: Complete substitution with NanoplasmidTM resulted in up to a 10-fold increase in vector genome titre relative to conventional plasmids under matched conditions. Hybrid systems failed to recapitulate this improvement, demonstrating that full-system backbone minimisation is required to ensure high yield. Productivity gains were preserved across transfection reagents and suspension media. NanoplasmidTM deployment represents a scalable, capsid-independent upstream intensification approach that improves yield without altering capsid biology. Conclusions: Integration of this approach within a design-for-manufacturability framework offers a practical route to reducing bacterial plasmid elements increasing safety, enhancing process robustness, and improving economic feasibility of AAV therapeutics.<\/jats:p>","DOI":"10.3390\/pharmaceutics18070814","type":"journal-article","created":{"date-parts":[[2026,7,1]],"date-time":"2026-07-01T03:06:34Z","timestamp":1782875194000},"page":"814","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":0,"title":["Backbone-Minimised Nanoplasmid DNA Systems Enable High-Titre AAV Production in Suspension HEK293 Platforms"],"prefix":"10.3390","volume":"18","author":[{"ORCID":"https:\/\/orcid.org\/0009-0007-5623-4665","authenticated-orcid":false,"given":"Lewis","family":"Hall","sequence":"first","affiliation":[{"name":"Department of Biochemical Engineering, University College London, London WC1E 6BT, UK"},{"name":"School of Pharmacy, University College London, London WC1N 1AX, UK"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Michael J.","family":"Fiore","sequence":"additional","affiliation":[{"name":"School of Pharmacy, University College London, London WC1N 1AX, UK"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Joel","family":"Abramovich","sequence":"additional","affiliation":[{"name":"School of Pharmacy, University College London, London WC1N 1AX, UK"},{"name":"Biotherapeutics and Advanced Therapies, Science and Research Group, Medicines and Healthcare Products Regulatory Agency, Potters Bar EN6 3GQ, UK"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Claire","family":"Kerridge","sequence":"additional","affiliation":[{"name":"Biotherapeutics and Advanced Therapies, Science and Research Group, Medicines and Healthcare Products Regulatory Agency, Potters Bar EN6 3GQ, UK"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-0044-0949","authenticated-orcid":false,"given":"Ahad A.","family":"Rahim","sequence":"additional","affiliation":[{"name":"School of Pharmacy, University College London, London WC1N 1AX, UK"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Qasim A.","family":"Rafiq","sequence":"additional","affiliation":[{"name":"Department of Biochemical Engineering, University College London, London WC1E 6BT, UK"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-5986-6082","authenticated-orcid":false,"given":"Giulia","family":"Massaro","sequence":"additional","affiliation":[{"name":"School of Pharmacy, University College London, London WC1N 1AX, UK"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"1968","published-online":{"date-parts":[[2026,6,30]]},"reference":[{"key":"ref_1","doi-asserted-by":"crossref","first-page":"78","DOI":"10.1038\/s41392-024-01780-w","article-title":"Adeno-associated virus as a delivery vector for gene therapy of human diseases","volume":"9","author":"Wang","year":"2024","journal-title":"Signal Transduct. 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