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Modeling Pulmonary Fibrosis by AAV-Mediated Tgfβ1 Expression – a Proof of Concept Study for AAV-Based Disease Modeling and Riboswitch-Controlled Vector Production

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Modeling Pulmonary Fibrosis by AAV-Mediated Tgfβ1 Expression – a Proof of Concept Study for AAV-Based Disease Modeling and Riboswitch-Controlled Vector Production Modeling pulmonary fibrosis by AAV-mediated TGFβ1 expression – a proof of concept study for AAV-based disease modeling and riboswitch-controlled vector production Dissertation submitted for the degree of Doctor of Natural Sciences (Dr. rer. nat.) Presented by Benjamin Strobel at the Faculty of Sciences Department of Biology Date of the oral examination: 07.04.2016 First referee: Prof. Dr. Florian Gantner Second referee: Prof. Dr. Marcel Leist Third referee: Prof. Dr. Jörg Hartig Konstanzer Online-Publikations-System (KOPS) URL: http://nbn-resolving.de/urn:nbn:de:bsz:352-0-332128 Parts of this thesis are published in: Strobel B , Klauser B, Hartig JS, Lamla T, Gantner F, Kreuz S. Riboswitch-mediated Attenuation of Transgene Cytotoxicity Increases Adeno-associated Virus Vector Yields in HEK-293 Cells. Mol Ther. 2015 Oct;23(10):1582-91. doi: 10.1038/mt.2015.123. Strobel B , Duechs MJ, Schmid R, Stierstorfer BE, Bucher H, Quast K, Stiller D, Hildebrandt T, Mennerich D, Gantner F, Erb KJ, Kreuz S. Modeling Pulmonary Disease Pathways Using Recombinant Adeno-Associated Virus 6.2. Am J Respir Cell Mol Biol. 2015 Sep;53(3):291-302. doi: 10.1165/rcmb.2014-0338MA. Reprinted with permission of the American Thoracic Society. Copyright © 2015 American Thoracic Society. The American Journal of Respiratory Cell and Molecular Biology is an official journal of the American Thoracic Society. Strobel B *, Miller FD*, Rist W, Lamla T. Comparative Analysis of Cesium Chloride- and Iodixanol- Based Purification of Recombinant Adeno-Associated Viral Vectors for Preclinical Applications. Hum Gene Ther Methods. 2015 Aug;26(4):147-57. doi: 10.1089/hgtb.2015.051. *equal contribution Parts of this thesis were presented at following scientific meetings: American Society for Gene and Cell Therapy (ASGCT) 18 th Annual Meeting, 2015, New Orleans. “Increasing AAV vector yield by riboswitch-mediated attenuation of toxic transgene effects in HEK-293 producer cells” (oral presentation). Strobel B , Klauser B, Lamla T, Gantner F, Kreuz S. European Respiratory Society (ERS) International Congress, 2014, Munich. “AAV-Tgfb1 vs. Bleomycin: Analysis of gene expression profiles in two models of pulmonary fibrosis” (oral presentation). Strobel B , Léparc G, Lämmle B, Hildebrandt T, Stierstorfer BE, Lamla T, Kreuz S. Gesellschaft für Virologie (GfV), 9 th workshop: Viral Vectors and Gene Therapy, 2014, Ulm. “Pulmonary disease modeling using AAV vectors” (oral presentation). Strobel B . Table of contents I SUMMARY iii II ZUSAMMENFASSUNG iv 1 INTRODUCTION 1 1.1 ADENO -ASSOCIATED VIRUS (AAV) VECTORS 2 1.1.1 AAV BIOLOGY 2 1.1.2 GENERATION OF RECOMBINANT AAV VECTORS 4 1.1.3 AAV CAPSID ENGINEERING FOR PRECLINICAL AND CLINICAL APPLICATIONS 7 1.1.4 APPLICATIONS OF AAV VECTORS IN RESPIRATORY RESEARCH 8 1.2 PULMONARY FIBROSIS 9 1.2.1 EPIDEMIOLOGY , CLINICAL ASPECTS AND PATHOBIOLOGY 9 1.2.2 TRANSFORMING GROWTH FACTOR β1 (TGF β1) AND ITS ROLE IN PULMONARY FIBROSIS 13 1.2.3 ANIMAL MODELS OF PULMONARY FIBROSIS 18 1.3 AIM OF THE THESIS 21 2 RESULTS 23 2.1 EVALUATION OF AAV6.2 AS A TOOL FOR LUNG GENE TRANSFER 24 2.1.1 ANALYSIS OF THE TRANSDUCTION PROFILE OF AAV6.2 24 2.1.2 IMMUNOLOGICAL PROFILE AND LONG -TERM TRANSGENE EXPRESSION 26 2.2 MODELING PULMONARY FIBROSIS BY AAV6.2-MEDIATED TGF β1 EXPRESSION 30 2.2.1 CONSTRUCT DESIGN AND VALIDATION OF TGF β1 BIOACTIVITY 30 2.2.2 TGF β1-INDUCED PATHOPHENOTYPE (DOSE -RESPONSE -RELATIONSHIP ) 33 2.2.3 AAV-TGF β1- VS . BLEOMYCIN -INDUCED FIBROSIS : COMPARATIVE ANALYSIS OF THE DISEASE COURSE AND GENE EXPRESSION PROFILES 37 2.2.4 PHARMACOLOGICAL INTERVENTION 58 2.3 RIBOSWITCHES AS NOVEL TOOLS TO REGULATE AAV TRANSGENE EXPRESSION 61 2.3.1 RIBOSWITCH -MEDIATED CONTROL OF TRANSGENE EXPRESSION IN HEK-293 CELLS 62 2.3.2 RIBOSWITCH -MEDIATED INCREASE IN AAV VECTOR YIELD 66 2.3.3 RIBOSWITCH -MEDIATED CONTROL OF AAV-MEDIATED TRANSGENE EXPRESSION 71 3 DISCUSSION 75 3.1 AAV AS A TOOL FOR DISEASE MODELING AND DRUG DISCOVERY RESEARCH 76 3.2 AAV-TGF β1- AND BLEOMYCIN-INDUCED PULMONARY FIBROSIS 79 3.3 CURRENT AND FUTURE APPLICATIONS OF AAV-RIBOSWITCH VECTORS 86 3.4 CONCLUSION 89 i 4 MATERIALS & METHODS 91 4.1 EQUIPMENT , CHEMICALS AND CONSUMABLES 92 4.2 MOLECULAR BIOLOGIC , CELL BIOLOGIC AND BIOCHEMICAL METHODS 94 4.2.1 CELL CULTURE 94 4.2.2 TRANSFECTION AND TRANSDUCTION OF CELLS 95 4.2.3 CLONING OF EXPRESSION CONSTRUCTS 95 4.2.4 EXPRESSION CONSTRUCTS 96 4.2.5 AAV PRODUCTION AND PURIFICATION 98 4.2.6 AAV GENOMIC TITER DETERMINATION 101 4.2.7 RNA PREPARATION FROM CELLS AND LUNG TISSUE 101 4.2.8 GENE EXPRESSION ANALYSIS BY Q PCR 102 4.2.9 RIBOSWITCH GFP ASSAY 103 4.2.10 FIBROBLAST -TO -MYOFIBROBLAST (FMT) ASSAY 104 4.2.11 CYTOTOXICITY ASSAY 105 4.2.12 LUNG IMMUNE CELL ANALYSIS 105 4.2.13 TOTAL PROTEIN MEASUREMENT (B RADFORD ASSAY ) 105 4.2.14 PROTEIN DETECTION USING ELISA 106 4.2.15 PROTEIN DETECTION USING WESTERN BLOT 106 4.2.16 HISTOLOGICAL EXAMINATION 106 4.2.17 ASHCROFT SCORING 107 4.3 IN VIVO METHODS AND ANIMAL HANDLING 107 4.3.1 ANIMALS 107 4.3.2 VIRAL VECTOR /SUBSTANCE ADMINISTRATION AND SAMPLING 108 4.3.3 LUNG FUNCTION MEASUREMENT 108 4.3.4 MICRO -CT ANALYSIS 108 4.4 NEXT GENERATION SEQUENCING , BIOINFORMATICS AND STATISTICS 109 4.4.1 LIBRARY PREPARATION 109 4.4.2 COMPUTATIONAL ANALYSIS 110 4.4.3 STATISTICS 111 III REFERENCES 112 IV LIST OF FIGURES AND TABLES v V RECORD OF CONTRIBUTIONS vii VI LIST OF ABBREVIATIONS viii VII DANKSAGUNG ix ii I Summary Adeno-associated virus (AAV) vectors have gained considerable attention as tools for the genetic manipulation of various cell types, tissues and organs in vitro and in vivo , which is facilitated by their non-pathogenicity, weak immunogenicity, low biosafety requirements (biosafety level S1) and comparatively easy recombinant production. However, despite the identification of AAV6.2, a capsid protein VP1 F129L point-mutated variant of AAV6, which showed superior transduction efficiency to other serotypes in the lung of mice, no studies have utilized AAVs for pulmonary disease modeling so far. To investigate AAV6.2’s suitability for this purpose, first, its pulmonary transduction efficiency and cellular tropism, transgene expression stability and immunogenicity were studied. Our results demonstrated that AAV6.2 enables broad transduction of the murine lung with stable transgene expression observed in bronchial epithelial and type II alveolar epithelial cells over the full tested time period of four months. Notably, in contrast to a commonly used Adenovirus-5 vector, AAV6.2 did not induce any measureable acute inflammation upon intratracheal administration, thereby lowering the risk of altering relevant readouts by vector- mediated immune responses. Moreover, AAV6.2-mediated overexpression of TGFβ1 dose- dependently induced pulmonary fibrosis in mice, which mirrored several key features of the human pathology, thereby establishing proof-of-concept for AAV-mediated disease modeling. A time course experiment in direct comparison to Bleomycin-induced lung fibrosis – the most widely used model of pulmonary fibrosis – identified key differences and commonalities in disease onset and progression. We found that the most distinct difference between both models lays in an acute injury/inflammation response in the Bleomycin model prior to fibrosis development, as opposed to a phase of relatively simultaneously occurring fibrosis and inflammation in the AAV model. By next generation sequencing, mRNA and microRNA expression changes were tracked and used to identify common disease signatures during fibrosis onset and maintenance. Utilizing this approach and correlation computation using lung functional changes, protein and miRNA candidates were identified, which might present attractive candidates for fibrosis drug discovery research, among them lysyl oxidase-like 2 (LOXL2), hyaluronan synthase 2 (HAS2), fibromodulin (FMOD) as well as miR-181a-5p, miR-676-3p and miR-192-5p. Furthermore, suitability of the AAV6.2-TGFβ1 model for compound testing was also established by pharmacological intervention using a type I TGFβ receptor inhibitor. Finally, to facilitate production of AAV vectors independent of the transgene used, we developed a novel gene regulation system based on an AAV-integrated artificial, self-cleaving, guanine-responsive riboswitch. This approach enabled efficient production of high-titer AAV vectors with close-to-normal in vivo bioactivity, and further enabled dynamic transgene expression control after AAV transduction in vitro . Following further engineering, such vectors might be ultimately used as gene expression control devices in AAV gene therapy. iii II Zusammenfassung Aufgrund der schwachen Immunogenität, geringen Biosicherheitsanforderungen (Sicherheitsstufe S1) und der verhältnismäßig einfachen rekombinanten Herstellung, sind die als nicht-pathogen geltenden Adeno-assoziierten Virus (AAV)-Vektoren weit verbreitet, um genetisches Material in verschiedene Zellen, Gewebe und Organe in vitro und in vivo einzubringen. Durch eine F129L Mutation in Kapsidprotein VP1 von AAV6, wurde eine AAV-Variante namens AAV6.2 generiert, die die bislang beste Lungen-Transduktionseffizienz in Mäusen aufwies. Jedoch gibt es trotz dieses positiven Ergebnisses bislang keine Lungenkrankheits-assoziierten Mausmodelle, die durch AAV- vermittelten Gentransfer erzeugt wurden. Um die Eignung von AAV6.2 Vektoren für diese Zwecke zu untersuchen, wurde zunächst deren Immunogenität, Transduktionseffizienz, ihr zellulärer Tropismus sowie die Stabilität der Transgenexpression untersucht. Hierbei konnte eine weitreichende Transduktion
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