Abstract

Especially in gene therapy applications, certain sequence motifs contained in plasmid DNA have to be avoided wherever possible. Such sequences are e.g. the bacterial ori or selection markers, only used for controlling the bacterial replication of the plasmid or to select for the plasmid during cloning or during production. Such sequence motifs are redundant in the intended therapeutic application and are completely removed in minicircles, i.e. circular and ccc-supercoiled expression cassettes.Since Adeno-Associated viral (AAV) vectors are produced by co-transfection of HEK293 producer cells, such bacterial sequence motifs may be an issue of an AAV vector-mediated gene transfer as well. Here, as a result of so-called reverse packaging events, an AAV vector-mediated transfer of not only the therapeutic gene but also of the antibiotic resistance gene into the target cells has been reported. Hence, this appears to be a potential risk of plasmid derived AAV vectors which can be overcome by using minicircle DNA for AAV production.Here we present first results showing that both constructs, the Helper & Packaging plasmid as well as the Transfer plasmid can be produced as minicircles although certain sequence motifs such as the ITRs have been identified to be an issue in minicircle production which has been overcome. These minicircles have been used for efficient AAV vector productions. However, only by replacing both, the Helper & Packaging plasmid as well as the transfer plasmid, encapsidation of the antibiotics resistance gene can be avoided.

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