DNA Minicircle Technology Improves Purity of Adeno-associated Viral Vector Preparations

Molecular Therapy: Nucleic Acids

Maria Schnödt¹  Laura Escalona Espinosa¹  Hildegard Büning¹  Anja Grünert²  Thomas Fuchsluger²  Barbara Kracher³  Christa Krüsemann⁴  Martin Schleef⁴  Marco Schmeer⁴  Anja Rischmüller⁴ 

[1] Center for Molecular Medicine Cologne (CMMC), University of Cologne, Cologne, Germany;Clinic for Ophthalmology, University Hospital Erlangen, Erlangen, Germany;Department of Plant-Microbe Interactions, Max-Planck Institute for Plant Breeding Research, Cologne, Germany;Plasmid Factory, Bielefeld, Germany;

关键词: AAV vectors;    DNA impurities;    Minicircle;    Vector production;    Vector quality;   

DOI  :  10.1038/mtna.2016.60

来源: DOAJ

【 摘 要 】

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.

【 授权许可】

Unknown