pIR-a new antibiotic free plasmid production platform for non-viral DNA delivery

Plasmid-based non-viral DNA delivery systems are widely utilized in gene therapy and cancer immunotherapy due to their safety and scalability. However, conventional production depends on antibiotic resistance markers, creating regulatory hurdles and complicating manufacturing. To overcome these limitations, we developed pIR, an antibiotic-free plasmid production platform that employs an arabinose-inducible Cre recombinase system to excise the antibiotic resistance gene during amplification. This system uses a pir+ bacterial strain supporting replication of R6K-origin plasmids, enabling high-yield production of antibiotic-free DNA suitable for clinical applications. We first verified that pIR generates high-purity DNA with over 80% of the final product in supercoiled form. Next, we compared pIR-derived plasmids to the traditional gWiz vector using Firefly luciferase (Fluc) and NanoLuc reporters in both in vitro and in vivo models. Our plasmids exhibited superior transgene expression and favorable safety profiles. Further comparison with the commercially available Nanoplasmid™ NTC9385R revealed comparable performance, with slightly higher NanoLuc expression in vitro and in vivo. To demonstrate versatility, we applied pIR to produce a plasmid encoding LINE1 ORF1p, a transposable element protein implicated in aging and cancer that constitutes ~20% of the human genome. In vitro studies confirmed robust ORF1p expression, and in vivo immunization via electroporation elicited a significantly stronger immune response compared to gWiz. By eliminating antibiotic sequences, pIR reduces the risk of horizontal gene transfer and aligns with regulatory requirements for clinical-grade DNA. Its ability to produce high-performance plasmids for both reporter and immunogenic payloads underscores its potential for gene therapy and cancer vaccine development. As non-viral DNA delivery gains momentum in oncology and personalized medicine, platforms that combine safety, efficiency, and compliance will be essential for accelerating translation. In conclusion, we establish pIR as an alternative to existing technologies, offering broad applications in cancer immunotherapy and beyond.