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

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 (EON and EOFF) 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-K1, 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.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.An array of different viral transduction systems are being used currently in pre-clinical and clinical trials [1-3]. Among these, vectors based on the replication-defective adeno-associated virus type 2 have attracted special attention as tools for clinical gene transfer. Different characteristics, such as (i) the ability to transduce dividing as well as non-dividing cells, (ii) high transduction rates in a wide range of tissues, and notably, (iii) the unique safety properties, make AAVs a promising vector in gene therapy initiatives [4-10].Over the pa