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Respiratory Syncytial Virus Based Vectors for the Treatment of Cystic Fibrosis DISSERTATION Presented in Partial Fulfillment Of

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Respiratory Syncytial Virus Based Vectors for the Treatment of Cystic Fibrosis DISSERTATION Presented in Partial Fulfillment Of Respiratory Syncytial Virus Based Vectors for the Treatment of Cystic Fibrosis DISSERTATION Presented in Partial Fulfillment of the Requirements for the Degree Doctor of Philosophy in the Graduate School of The Ohio State University By Anna R. Kwilas The Integrated Biomedical Science Graduate Program The Ohio State University 2010 Dissertation Committee: Mark Peeples Douglas McCarty Joanne Trgovcich Michael Oglesbee Copyright by Anna R. Kwilas 2010 Abstract Cystic fibrosis (CF) is the most common lethal recessive genetic disease in the Caucasian population. It is caused mutations in the CF transmembrane conductance regulator (CFTR) gene which lead to the production of a partially or completely non-functional protein. Lack of CFTR in epithelial cell membranes results in abnormal ion transport and dehydration of mucosal surfaces. In the respiratory tract this leads severe lung disease, the leading cause of morbidity and mortality in the CF population. Since CFTR was first cloned in 1989, a major goal of CF research has been to develop effective gene therapy for CF lung disease. Respiratory syncytial virus (RSV) naturally infects the ciliated cells that line the human respiratory tract, cells that express CFTR and play a major role in determining the hydration of the airway mucosal surface. In addition, although an immune response is mounted against RSV, this does not prevent subsequent infections suggesting that an RSV-based vector might be effectively re-administered a requirement since the apical ciliated cells of the respiratory tract are terminally differentiated and have a defined life span. To test whether the large, 4.5 kb, CFTR gene could be expressed by a recombinant RSV and whether infectious virus could be used to deliver CFTR to ciliated airway epithelium derived from CF patients, we have inserted the CFTR gene into four sites in the recombinant, green fluorescent protein-expressing (rg)RSV genome to generate virus expressing four different amounts of the CFTR protein. Two of these rgRSV-CFTR viruses were capable of expressing CFTR with little effect on viral replication. rgRSV-delivered CFTR was functional in primary CF airway epithelial cultures. Infection with these viruses resulted in chloride channel ii function similar to that seen in non-CF cultures and correction of additional characteristic defects seen in CF airway epithelia. Unfortunately, most ciliated airway cells infected with RSV die within 5 days. To create an improved RSV-based vector we generated an RSV “replicon” by removing the three glycoprotein genes from a full-length RSV cDNA. The replicon is able to replicate in cells without killing them or producing infectious virus, allowing the isolation and expansion of replicon-containing cells. Providing the viral glycoproteins in trans enables the production of “one-step virus” (OSV) which is capable of delivering a replicon to fresh cells where it produces its encoded proteins, but again cannot spread. We inserted the CFTR gene into the RSV replicon genome in four positions. All of these replicons were capable of producing the CFTR protein. When CFTR-expressing replicon OSV were used to infect primary human airway epithelial cultures, replicon containing cells remained within the cultures for 12-20 days, indicating that replicon containing cells have a survival advantage over those infected with complete virus. Our results indicate that RSV is capable of expressing CFTR, supplying enough functional CFTR to primary CF airway cells to correct their physiologic defects and with modification can remain in primary airway cells for a prolonged period of time. Collectively, our data support further investigation of RSV as a potential vector for CF gene therapy. iii Dedication This work is dedicated to my family. To my mother, Judy, for providing me with an amazing role model, for always believing in me and for supporting me on all my decisions in life. To my father, Bob, for always being there for me, no matter where, no matter what time, and for always knowing exactly what to say or do. To my husband. Steve, for being my rock, for understanding me when no one else could and for being the love of my life. I would also like to dedicate this work to the father of the Integrated Biomedical Graduate Program, Dr. Allan Yates (1943-2010). His passion for knowledge and his ability to motivate students to achieve their best is unmatchable and he will surely be missed. iv Acknowledgments Thank you to the General Mason Scholarship Fund of The Research Institute at Nationwide Children’s Hospital for funding my graduate work. Thank you to Dr. Mark Peeples for all his guidance and support in my scientific career. Thanks to all the members of the Peeples’ laboratory particularly Barb Newton, Steve Kwilas, Arife Unal and Katelyn Leahy for their practical assistance. Thank you to John Riordan at UNC for the monoclonal antibody to CFTR and to everyone in the Pickles lab at UNC for their assistance with CFTR functional testing. I would also like to thank the members of my Dissertation Committee for all of their personal and professional help throughout my graduate career. I can’t imagine a better set of mentors. Portions of this document are Copyright © American Society for Microbiology, Journal of Virology, 2010. 84(15): p. 7770-81. v Vita 1998 ...................................................................................... Albert Gallatin Senior High School 2002 ...................................................................................... B.S. Animal Science, West Virginia University 2003 to present .............................................................. Graduate Research Associate, Department of Medicine, The Ohio State University Publications Kwilas, A.R., et al., Respiratory syncytial virus engineered to express the cystic fibrosis transmembrane conductance regulator corrects the bioelectric phenotype of human cystic fibrosis airway epithelium in vitro. J Virol, 2010. 84(15): p. 7770-81. Fields of Study Major Field: Integrated Biomedical Science Minor Fields: Molecular Virology and Gene Therapy, Translational Research vi Table of Contents Abstract .............................................................................................................................................................................. ii Dedication ........................................................................................................................................................................ iv Acknowledgments ......................................................................................................................................................... v Vita ....................................................................................................................................................................................... vi List of Figures .............................................................................................................................................................. xiii Abbreviations .............................................................................................................................................................. xiv Chapter 1: Cystic Fibrosis .......................................................................................................................................... 1 History............................................................................................................................................................................ 1 Epidemiology .............................................................................................................................................................. 2 The Cystic Fibrosis Transmembrane Conductance Regulator ............................................................ 4 CFTR Biosynthesis .................................................................................................................................................... 6 CFTR as a Cl- Channel ........................................................................................................................................... 10 CFTR as a Conductance Regulator ................................................................................................................. 16 Additional CFTR Functions ............................................................................................................................... 20 Interactions of CFTR with Other Proteins ................................................................................................. 21 CFTR Mutaions in the Human Population ................................................................................................. 23 CF Disease Manifestations ................................................................................................................................. 27 vii Airways .................................................................................................................................................................. 28 Pancreas ................................................................................................................................................................ 40 Gut ............................................................................................................................................................................ 42 Liver and Biliary Network ...........................................................................................................................
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