Adeno-associated viral vectors engineered for macrolide-adjustable transgene expression In mammalian cells and mice

Background: Adjustable gene expression is crucial in a number of applications such as de- or transdifferentiation of cell phenotypes, tissue engineering, various production processes as well as gene-therapy initiatives. Viral vectors, based on the Adeno- Associated Virus (AAV) type 2, have emerged as one of the most promising types of vectors for therapeutic applications due to excellent transduction efficiencies of a broad variety of dividing and mitotically inert cell types and due to their unique safety features. Results: We designed recombinant adeno- associated virus (rAAV) vectors for the regulated expression of transgenes in different configurations. We integrated the macrolide-responsive E. REX systems (E-ON and E-OFF) into rAAV backbones and investigated the delivery and expression of intracellular as well as secreted transgenes for binary set-ups and for self-and auto-regulated one-vector configurations. Extensive quantitative analysis of an array of vectors revealed a high level of adjustability as well as tight transgene regulation with low levels of leaky expression, both crucial for therapeutical applications. We tested the performance of the different vectors in selected biotechnologically and therapeutically relevant cell types (CHO-KI, HT-1080, NHDF, MCF-7). Moreover, we investigated key characteristics of the systems, such as reversibility and adjustability to the regulating agent, to determine promising candidates for in vivo studies. To validate the functionality of delivery and regulation we performed in vivo studies by injecting particles, coding for compact self-regulated expression units, into mice and adjusting transgene expression. Conclusion: Capitalizing on established safety features and a track record of high transduction efficiencies of mammalian cells, adeno-associated virus type 2 were successfully engineered to provide new powerful tools for macrolide-adjustable transgene expression in mammalian cells as well as in mice.