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.

Plasmid DNA (pDNA) vectors are the current conventional technology driving therapeutic gene transfer, whether for use toward mal/nonfunctional gene replacement, DNA vaccination, or production of therapeutic proteins in mammalian cells. However, the conventional pDNA vector suffers from several safety and efficiency limitations: 1) it imparts adverse immune responses to bacterial sequences required for maintenance and amplification in prokaryotes; 2) its bioavailability can be compromised due to size; and 3) it may be genotoxic due to its potential to integrate into the host chromosome and yield an oncogenic event. In this study we have constructed a robust in vivo bacterial platform for the production of bacterial sequence-free linear covalently closed (LCC) DNA vectors, termed DNA Ministrings, through the manipulation and application of bacteriophage-encoded recombination systems. Phage N15 and PY54 lysogenize their bacterial hosts as a linear plasmid with covalently closed ends (LCC plasmid). LCC morphology is conferred by the phage-encoded telomerase via a single cleaving-joining reaction of the perfect palindrome target site. This system was exploited to generate DNA Ministring vectors, encoding only the gene(s) of interest and necessary complementary eukaryotic expression/enhancement genetic elements that are devoid of unwanted bacterial sequences and are linearized through a single in vivo enzymatic reaction. The tel and telN prokaryotic telomerase (protelomerase) genes were amplified from PY54 and N15 lysates, respectively, and cloned into a bacterial vector that expresses the gene under control of the temperature sensitive bacteriophage ?? CI857 repressor that confers conditional expression from ?? pL/pR promoters. This regulatory circuit was integrated into a RecA+ lacZ+ E. coli K-12 strain via homologous recombination, where successful recombinants were disrupted for the lacZ gene. Recombinant cells are capable of conditional expression of the phage-derived telomerase enzymes by shifting the temperature to >37 ??C. Phage P1-derived Cre recombinase was applied as a positive control, since its functionality in generating DNA minicircle vectors has been previously shown. A multi-purpose 342 bp target site termed Super Sequence (SS) that possesses the Cre, Flp, Tel, and TelN target sites in addition to two flanking SV40 enhancer sequences was cloned into two different sites of a GFP expression eukaryotic pDNA vector. The amplification of this DNA vector through telN / tel or cre expressing Recombinant E. coli cells (R-cells) generated bacterial sequence-depleted (LCC) DNA Ministring and (CCC) Minicircle vectors, respectively, as evidenced by digestion patterns of the purified vector. Transfection efficiency of these vectors was assessed in rapidly dividing human ovarian cancer and in relatively slowly dividing human embryonic kidney cell lines. In vitro experiments with DNA Ministrings in human cells lines resulted in significantly higher transfection efficiency, bioavailability, and cytoplasmic diffusion levels compared to the parental plasmid precursor and isogenic DNA Minicircle counterparts. The safety of the LCC DNA vector conformation, with respect to insertional genotoxicity, was assessed by forcing LCC pDNA vectors into bacterial and human genomic DNA. The integration of LCC DNA into bacterial and human host genomic DNA resulted in chromosomal DNA disruptions at site of integration, loss of genome stability, and subsequent cell death. LCC integration-induced apoptotic cell death and natural elimination of the integrant from human cell population improves the safety profile of DNA Ministrings by eliminating integrants following the potential genotoxic side effects of undesired vector integration into the host genome.