Adeno-associated viral (AAV) vectors have emerged as one of the most popular gene transfer systems in both research and clinical gene therapy. As AAV vectors are derived from a stealth, nonpathogenic virus and lack active integrase activity, these vectors are frequently applied for in vivo gene therapy of liver, muscle, and other postmitotic tissues. Although long-term transgene expression from AAV vector episomes is reported from these tissues, the episomal nature of AAV-once regarded as disadvantage-has become an attractive feature for gene-editing approaches targeting proliferating cells. In response to the high demand, AAV vector production is receiving special attention. Besides particle yields and biological activity, the most important concern is improving vector purity. The most difficult task in this regard is removal of defective particles, that is, capsids that are either empty or contain DNA other than the full-length vector genomes. Herein, we characterize and discuss these so-called product-related impurities, methods for their detection, as well as strategies to avoid or reduce their formation.

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.

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