Optimized Production and Purification of LCC DNA Minivectors for Applications in Gene Therapy and Vaccine Development

Abstract

Linear covalently closed (LCC) DNA minivectors serve to be superior to conventional circular covalently closed (CCC) plasmid DNA (pDNA) vectors due to enhancements to both transfection efficiency and safety. Specifically, LCC DNA minivectors have a heightened safety profile as insertional mutagenesis is inhibited by covalently closed terminal ends conferring double-strand breaks that cause chromosomal disruption and cell death in the low frequency event of chromosomal integration. The development of a one-step, E. coli based in vivo LCC DNA minivector production system enables facile and efficient production of LCC DNA minivectors referred to as DNA ministrings. This novel in vivo system demonstrates high versatility, generating DNA ministrings catered to numerous potential applications in gene therapy and vaccine development. In the present study, numerous aspects pertaining to the generation of gene therapeutics with LCC DNA ministrings have been explored with relevance to both industry and clinical settings. Through systematic assessment of induction duration, cultivation strategy, and genetic/chemical modifications, the novel in vivo system was optimized to produce high yields of DNA ministrings at ~90% production efficiency. Purification of LCC DNA ministrings using anion exchange membrane chromatography demonstrated rapid, scalable purification of DNA vectors as well as its potential in the separation of different DNA isoforms. The application of a hydrogel-based strong Q-anion exchange membrane, with manipulations to salt gradient, constituted effective separation of parental supercoiled CCC precursor pDNA and LCC DNA. The resulting DNA ministrings were employed for the generation of 16-3-16 gemini surfactant based synthetic vectors and comparative analysis, through physical characterization and in vitro transfection assays, was conducted between DNA ministring derived and CCC pDNA derived lipoplexes. Differences in DNA topology were observed to induce differences in particle size and DNA protection/encapsulation upon lipoplex formation. Lastly, the in vivo DNA minivector production system successfully generated gagV3(BCE) LCC DNA ministrings for downstream development of a HIV DNA-VLP (Virus-like particle) vaccine, thus highlighting the capacity of such system to produce DNA ministrings with numerous potential applications.