Universität zu Köln

Schnoedt, Maria, Schmeer, Marco, Kracher, Barbara, Kruesemann, Christa, Espinosa, Laura Escalona, Gruenert, Anja, Fuchsluger, Thomas, Rischmueller, Anja, Schleef, Martin and Buening, Hildegard (2016). DNA Minicircle Technology Improves Purity of Adeno-associated Viral Vector Preparations. Mol. Ther.-Nucl. Acids, 5. CAMBRIDGE: CELL PRESS. ISSN 2162-2531

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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.

Item Type:	Journal Article
Creators:	Creators Email ORCID ORCID Put Code Schnoedt, Maria UNSPECIFIED UNSPECIFIED UNSPECIFIED Schmeer, Marco UNSPECIFIED UNSPECIFIED UNSPECIFIED Kracher, Barbara UNSPECIFIED UNSPECIFIED UNSPECIFIED Kruesemann, Christa UNSPECIFIED UNSPECIFIED UNSPECIFIED Espinosa, Laura Escalona UNSPECIFIED UNSPECIFIED UNSPECIFIED Gruenert, Anja UNSPECIFIED UNSPECIFIED UNSPECIFIED Fuchsluger, Thomas UNSPECIFIED UNSPECIFIED UNSPECIFIED Rischmueller, Anja UNSPECIFIED UNSPECIFIED UNSPECIFIED Schleef, Martin UNSPECIFIED UNSPECIFIED UNSPECIFIED Buening, Hildegard UNSPECIFIED UNSPECIFIED UNSPECIFIED
URN:	urn:nbn:de:hbz:38-266358
DOI:	10.1038/mtna.016.60
Journal or Publication Title:	Mol. Ther.-Nucl. Acids
Volume:	5
Date:	2016
Publisher:	CELL PRESS
Place of Publication:	CAMBRIDGE
ISSN:	2162-2531
Language:	English
Faculty:	Unspecified
Divisions:	Unspecified
Subjects:	no entry
Uncontrolled Keywords:	Keywords Language VIRUS TYPE-2; GENE-THERAPY; TRANSGENE EXPRESSION; BACTERIAL-DNA; RECOMBINANT PLASMIDS; TERMINAL REPEATS; AAV VECTORS; RESCUE; PURIFICATION; REPLICATION Multiple languages Medicine, Research & Experimental Multiple languages
Refereed:	Yes
URI:	http://kups.ub.uni-koeln.de/id/eprint/26635