1. Optimization of adenoviral transduction of the human muscle tissue for the development of ex vivo gene therapy Vesna Vetma 1, Maja Antunović 1, Igor Matić 1, Eduard Rod 2, Alan Ivković 2, Inga Marijanović 1 1 Department of Molecular Biology, Faculty of Science, University of Zagreb, Zagreb, Croatia ( inga. marijanovic @biol.pmf.hr), inga. marijanovic @biol.pmf.hr inga. marijanovic @biol.pmf.hr 2 Department of Orthopaedics, Clinical Hospital “Sveti Duh”, Zagreb, Croatia The need to improve bone healing permeates the discipline of orthopedic surgery. Osteogenic inducers must be available for sufficient period of time and in sufficient amount to promote osteogenesis. One approach to achieving this goal is gene therapy. Using adenoviral gene transfer it is possible to create human skeletal muscle cells that produce osteogenic inducers and subsequently return those cells at the place of healing. It is essential that gene therapy is “same day“ ex vivo regional gene therapy, with the surgery done within two hours. Introduction Results Conclusions Aim To optimize the transduction of grafts of human muscle with a replication defective adenoviral vector constructed to carry the luciferase reporter gene (Ad.Luc 2) in vitro. The transduction efficiency of adenoviral vector in vitro will depend on: a) viral titer, b) period of contact with virus and c) presence of calcium and/or lanthanum ions. Adenoviral vector expressing osteogenic inducer BMP-2 (Bone Morphogenetic Protein) will be used to evaluate the effectiveness of the developed protocol. Ideal viral titer for transduction of human muscle graft was 10 9 PFU/mL. The ideal period of contact between muscle graft and adenovirus was 30 minutes and there was no influence of La and Ca ions on the effectiveness of transduction, probably due to the already high viral titer. The effectiveness of the protocol was prosperous according to qPCR analysis. Materials and methods The ideal concentration of adenoviral particles, transduction time and influence of lanthanum chloride and/or calcium chloride on improvement of transduction was determined by luciferase report assay and the BCA protein assay. AdBMP-2 was used to evaluate the effectiveness of developed protocol. Quantitative PCR was done on day 0, 7 and 14 to confirm the expression of DMP1 and BSP, which encode for bone extracellular matrix proteins. Osteogenic induction medium ( α MEM 10% FBS + P/S + 50 μ g/ml ascorbic acid + 4 mM β -glycerophosphate + 1 μ M dexamethasone) was used as expected inductor of expression of DMP1 and BSP. Figure 1. Figure 1. The ideal concentration of adenoviral particles for transduction of human muscle graft measured by luciferase report assay and BCA protein assay 72h after transduction. Human muscle grafts were treated with 0, 10 6, 10 7, 10 8 and 10 9 PFU/mL adenoviral titer for 2 hours. 10 9 PFU/mL was the best viral titer for transduction of muscle grafts. Figure 2. Figure 2. The ideal period of contact between muscle graft and adenoviral vector measured by luciferase report assay and BCA protein assay 72h after transduction. Viral titer applied on the muscle graft was 109 PFU/mL according to the determined ideal concentration for transduction. Muscle graft was exposed to specific viral concentration for 0, 15, 30 or 60 minutes. The ideal time of contact between muscle graft and adenovirus is 30 minutes, since there was no statistically significant difference between 30 and 60 minutes of exposure. Figure 3. Figure 3. The effect of lanthanum and calcium ions on improvement of transduction of human muscle graft was measured by firefly luciferase report assay and BCA protein assay 72h after transduction. Human muscle grafts were treated with 10 9 PFU/mL viral titer and 5mM CaCl2 or 0.2mM LaCl3 or both for 30 minutes. There was no statistically significant difference between pure viral titer applied and supplemented viral titer applied to the muscle grafts. Figure 4. Figure 4. Relative mRNA expression of BSP and DMP1 on day0, day7 and day14. Human muscle grafts were treated with 10 9 PFU/mL AdBMP-1for 30 min, without La and Ca and RNA was isolated on day0, 7 and 14. The highest expression was observed for muscle graft treated with AdBMP-1 and osteogenic midium as expected, but significant increase of mRNA expression of both genes was as well for muscle graft treated with AdBMP-1on the day14.