title: Minicircle DNA immobilized in bacterial ghosts: in vivo production of safe non-viral DNA delivery vehicles.
abstract: DNA as an active agent is among the most promising technologies for vaccination and therapy. However, plasmid backbone sequences needed for the production of pDNA in bacteria are dispensable, reduce the efficiency of the DNA agent and, most importantly, represent a biological safety risk. In this report we describe a novel technique where a site-specific recombination system based on the ParA resolvase was applied to a self-immobilizing plasmid system (SIP). In addition, this system was combined with the protein E-specific lysis technology to produce non-living bacterial carrier vehicles loaded with minicircle DNA. The in vivo recombination process completely divided an origin plasmid into a minicircle and a miniplasmid. The replicative miniplasmid containing the origin of replication and the antibiotic resistance gene was lost during the subsequently induced PhiX174 gene E-mediated lysis process, which results in bacterial ghosts. The minicircle DNA was retained in these empty bacterial cell envelopes during the lysis process via the specific interaction of a membrane anchored protein with the minicircle DNA. Using this novel platform technology, a DNA delivery vehicle--consisting of a safe bacterial carrier with known adjuvant properties and minicircle DNA with an optimized safety profile--can be produced in vivo in a continuous process. Furthermore, this study provides the basis for the development of an efficient in vitro minicircle purification process.
authors.affiliation: Institute of Microbiology and Genetics, Section Microbiology and Biotechnology, Veterinary Medicine, University of Vienna, Veterinärplatz 1, A-1210 Vienna, Austria. Wolfgang.Jechlinger@vu-wien.ac.at
authors.forename: Wolfgang
authors.initials: W
authors.lastname: Jechlinger
authors.forename: Chakameh
authors.initials: C
authors.lastname: Azimpour Tabrizi
authors.forename: Werner
authors.initials: W
authors.lastname: Lubitz
authors.forename: Peter
authors.initials: P
authors.lastname: Mayrhofer
mesh_terms: D001419:Bacteria / Q000235:genetics*; D004269:DNA, Bacterial / Q000235:genetics / Q000378:metabolism; D004270:DNA, Circular / Q000235:genetics* / Q000378:metabolism*; D004274:DNA, Recombinant / Q000235:genetics / Q000378:metabolism; D015316:Genetic Therapy / Q000379:methods*; D005822:Genetic Vectors; D010957:Plasmids / Q000235:genetics / Q000378:metabolism; D045522:Recombinases / Q000235:genetics / Q000502:physiology; D011995:Recombination, Genetic; D019444:Vaccines, DNA; D014764:Viral Proteins / Q000235:genetics / Q000502:physiology
chemical_list: D004269:DNA, Bacterial; D004270:DNA, Circular; D004274:DNA, Recombinant; C029555:E protein, bacteriophage X174; D045522:Recombinases; D019444:Vaccines, DNA; D014764:Viral Proteins