title: DNA Minicircle Technology Improves Purity of Adeno-associated Viral Vector Preparations.
abstract: Adeno-associated viral (AAV) vectors are considered as one of the most promising delivery systems in human gene therapy. In addition, AAV vectors are frequently applied tools in preclinical and basic research. Despite this success, manufacturing pure AAV vector preparations remains a difficult task. While empty capsids can be removed from vector preparations owing to their lower density, state-of-the-art purification strategies as of yet failed to remove antibiotic resistance genes or other plasmid backbone sequences. Here, we report the development of minicircle (MC) constructs to replace AAV vector and helper plasmids for production of both, single-stranded (ss) and self-complementary (sc) AAV vectors. As bacterial backbone sequences are removed during MC production, encapsidation of prokaryotic plasmid backbone sequences is avoided. This is of particular importance for scAAV vector preparations, which contained an unproportionally high amount of plasmid backbone sequences (up to 26.1% versus up to 2.9% (ssAAV)). Replacing standard packaging plasmids by MC constructs not only allowed to reduce these contaminations below quantification limit, but in addition improved transduction efficiencies of scAAV preparations up to 30-fold. Thus, MC technology offers an easy to implement modification of standard AAV packaging protocols that significantly improves the quality of AAV vector preparations.
authors.affiliation: Center for Molecular Medicine Cologne (CMMC), University of Cologne, Cologne, Germany; German Center for Infection Research (DZIF), partner sites Bonn-Cologne and Hannover-Braunschweig, Germany; Department I of Internal Medicine, University Hospital Cologne, Cologne, Germany.
authors.forename: Maria
authors.initials: M
authors.lastname: Schnödt
authors.affiliation: Plasmid Factory, Bielefeld, Germany.
authors.forename: Marco
authors.initials: M
authors.lastname: Schmeer
authors.affiliation: Department of Plant-Microbe Interactions, Max-Planck Institute for Plant Breeding Research, Cologne, Germany.
authors.forename: Barbara
authors.initials: B
authors.lastname: Kracher
authors.affiliation: Plasmid Factory, Bielefeld, Germany.
authors.forename: Christa
authors.initials: C
authors.lastname: Krüsemann
authors.affiliation: Center for Molecular Medicine Cologne (CMMC), University of Cologne, Cologne, Germany; Department I of Internal Medicine, University Hospital Cologne, Cologne, Germany.
authors.forename: Laura Escalona
authors.initials: LE
authors.lastname: Espinosa
authors.affiliation: Clinic for Ophthalmology, University Hospital Erlangen, Erlangen, Germany.
authors.forename: Anja
authors.initials: A
authors.lastname: Grünert
authors.affiliation: Clinic for Ophthalmology, University Hospital Erlangen, Erlangen, Germany.
authors.forename: Thomas
authors.initials: T
authors.lastname: Fuchsluger
authors.affiliation: Plasmid Factory, Bielefeld, Germany.
authors.forename: Anja
authors.initials: A
authors.lastname: Rischmüller
authors.affiliation: Plasmid Factory, Bielefeld, Germany. Electronic address: Martin.schleef@plasmidfactory.com.
authors.forename: Martin
authors.initials: M
authors.lastname: Schleef
authors.affiliation: Center for Molecular Medicine Cologne (CMMC), University of Cologne, Cologne, Germany; German Center for Infection Research (DZIF), partner sites Bonn-Cologne and Hannover-Braunschweig, Germany; Department I of Internal Medicine, University Hospital Cologne, Cologne, Germany; Institute of Experimental Hematology, Hannover Medical School, Hannover, Germany. Electronic address: buening.hildegard@mh-hannover.de.
authors.forename: Hildegard
authors.initials: H
authors.lastname: Büning
keywords: AAV vectors; DNA impurities; Minicircle; Vector production; Vector quality