Targeting Transcription Factor NF-κB by Dual Functional Oligodeoxynucleotide Complex for Inhibition of Neuroinflammation A dissertation submitted to the Graduate School of the University of Cincinnati in partial fulfillment of the requirements for the degree of Doctor of Philosophy in the Department of Pharmaceutical Sciences of the College of Pharmacy by Jing Hu M.S. University of Cincinnati July 2015 Committee Chair: Jiukuan Hao, Ph.D. ABSTRACT Because transcription factor nuclear factor (NF)-κB plays an important role in vascular inflammation at blood-brain barrier, the present study constructed a novel DNA complex to specifically target endothelial NF-κB for inhibition of cerebral inflammation. This DNA complex (GS24-NFκB) contains two functional domains: a DNA Decoy for inhibiting NF-κB activity and a DNA aptamer (GS-24), a ligand of transferrin receptor (TfR), for targeted delivery of the DNA Decoy into brain endothelial cells. The results indicated that GS24-NFκB was delivered into murine brain-derived endothelial (bEnd5) cells and the delivery was specific and TfR-dependent. The DNA chimera inhibited inflammatory responses in the cells induced by tumor necrosis factor α (TNF-α) or oxygen-glucose deprivation/reoxygenation (OGD/R) via down-regulation of nuclear NF- κB subunit, P65, as well as its downstream inflammatory cytokines, such as inter cellular adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule (VCAM-1). The anti-inflammatory effect of the GS24-NFκB was shown as significant reduction in monocyte adhesion to the bEnd5 cells induced by TNF-α or OGD/R after GS24-NFκB treatment. Pharmacokinetic study suggested its poor nuclease resistance and rapid clearance from systemic circulation. Organ uptake of GS24-NFκB including brain uptake was much higher compared to the scramble control and high organ uptake was observed in liver and spleen with high expression of TfR. The brain uptake is 0.39%ID/g, which is comparable to the brain uptake of anti-TfR monoclonal antibody, OX26 (0.44%ID/g). Intravenous injection of GS24-NFκB (15mg/kg) was able to significantly reduce the expressions of phospho-P65 and VCAM-1 in brain endothelial cells in mouse ii lipopolysaccharide (LPS) induced inflammatory model, suggesting effective inhibition of activation and transcriptional activity of NF-κB. However, the disease-modifying effects of GS24-NFκB need to be further investigated in future. In conclusion, our approach using DNA nanotechnology has successfully delivered the therapeutic decoy into brain endothelial cells and induce pharmacological effect. It could be potentially applied for inhibition of inflammation in ischemic stroke and other neuroinflammatory diseases affecting cerebral vasculature. iii iv ACKNOWLEDGEMENTS First and foremost, I would like to thank my research advisor Dr.Jiukuan Hao for his guidance and support for my Ph.D. studies. I would like to thank him for welcoming me into his lab five years ago when I was a master and for giving me the opportunity to pursue a Ph.D. degree in University of Cincinnati. In Dr. Hao’s lab, I was exposed to diverse scientific disciplines and challenging but rewarding projects. He has encouraged me to present my work in many environments. I really appreciate his guidance and support which has truly pushed me to reach my full potential. I would like to acknowledge my committee members, Dr. Gary A. Gudelsky, Dr. Kevin Li, Dr. Kim B. Seroogy and Dr. Giovanni M. Pauletti, for their valuable advice for the project and their helpful discussions in committee meetings. In addition, I am very grateful to Dr. Pauletti. As the director of graduate program in pharmaceutical sciences, his help and friendship make me really enjoy the life in University of Cincinnati. I want to express my thanks to Dr. Seroogy and Dr. Ann M. Hemmerle as our collaborators for their help in establishing animal model and immunostaining. I also would like to thank Dr.Gudelsky for his kindness of sharing devices with us in animal experiments. I would like to thank Dr. Gerald Kasting and Dr. Allison Rush for helping me use their phosphorimager. I would like to thank Dr. Peixuan Guo and Dr. Yi Shu for their help in RNA synthesis. Many thanks to Dr.Yongguang Yang, Dr. Chaofeng Mu, Dr. He Wen, Dr. Linli Zhou, Daniah Alwaily, Yihong Deng, Xiaoxian An, Jiraganya Bhongsatierm and Hari Krishna Ananthula for strengthening and guiding me whenever I needed it. v I would like to thank every faculty member at the University of Cincinnati, who has ever taught and advised me. I would like to thank all the staff in college of pharmacy who has ever helped me. Lastly and most importantly, I would like to thank my family members: my husband Xu, my parents and in-laws, whose support made this happen. vi TABLE OF CONTENTS CHAPTER 1 INTRODUCTION ....................................................................................... 1 CHAPTER 2 REVIEW OF THE LITERATURE .............................................................. 6 2.1 Neuroinflammation ................................................................................................... 6 2.1.1 Blood-brain barrier ............................................................................................. 7 2.1.2 CECs-mediated inflammation ............................................................................ 8 2.2 NF-κB ...................................................................................................................... 10 2.3 Oligonucleotide therapy .......................................................................................... 14 2.3.1 Transcription factor decoy ................................................................................ 15 2.3.2 NF-κB decoy..................................................................................................... 16 2.4 Receptor-mediated brain delivery ........................................................................... 18 2.4.1 Transferrin receptor .......................................................................................... 19 2.5 Aptamer-mediated gene therapy ............................................................................. 21 2.5.1 Aptamers ........................................................................................................... 21 2.5.2 Aptamers in drug delivery ................................................................................ 22 2.5.3 Aptamer-therapeutic ODN conjugates ............................................................. 25 CHAPTER 3 AIMS OF THE STUDY ............................................................................. 29 CHAPTER 4 MATERIALS AND METHODS ............................................................... 30 4.1 Cell culture .............................................................................................................. 30 4.2 Animals ................................................................................................................... 30 4.3 Preparation of GS24-NFκB complex ...................................................................... 31 4.4 Cellular uptake of GS24-NFκB by bEnd5 cells ...................................................... 32 4.4.1 Flow cytometry ................................................................................................. 32 4.4.2 TfR (CD71) siRNA transfection....................................................................... 32 vii 4.4.3 Quantitative cellular uptake of 32P-labeled DNAs ........................................... 32 4.5 DNA treatments in inflammatory models in vitro .................................................. 33 4.5.1 TNF-α model .................................................................................................... 33 4.5.2 The OGD/R model............................................................................................ 33 4.5.3 Preparation of protein samples ......................................................................... 34 4.5.4 Western blot ...................................................................................................... 35 4.5.5 RT-PCR ............................................................................................................ 35 4.5.6 Adhesion assay ................................................................................................. 36 4.6 Pharmacokinetics and tissue distribution ................................................................ 37 4.7 DNA treatment in mouse LPS model ...................................................................... 38 4.7.1 DNA treatment in LPS model .......................................................................... 38 4.7.2 Brain microvessel isolation .............................................................................. 38 4.7.3 Sickness behavior analysis ............................................................................... 39 4.8 Statistical analysis ................................................................................................... 39 CHAPTER 5 RESULTS ................................................................................................... 40 5.1 Formation of GS24-NFκB complex ........................................................................ 40 5.2 Targeted delivery of GS24-NFκB into bEnd5 cells ................................................ 41 5.3 Pharmacological effect of GS24-NFκB in vitro ...................................................... 43 5.3.1 GS24-NFκB reduced nuclear P65