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.

We present a prototype nanofluidic device, developed for the continuous-flow dielectrophoretic (DEP) fractionation, purification, and quality control of sample suspensions for gene vaccine production. The device consists of a cross injector, two operation regions, and separate outlets where the analytes are collected. In each DEP operation region, an inhomogeneous electric field is generated at a channel spanning insulating ridge. The samples are driven by ac and dc voltages that generate a dielectrophoretic potential at the ridge as well as (linear) electrokinetics. Since the DEP potential differs at the two ridges, probes of three and more species can be iteratively fully fractionated. We demonstrate the fast and efficient separation of parental plasmid, miniplasmid, and minicircle DNA, where the latter is applicable as a gene vaccine. Since the present technique is virtually label-free, it offers a fast purification and in-process quality control with low consumption, in parallel, for the production of gene vaccines.

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