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Mrna Modification and Delivery Strategies Towards the Establishment of a Platform for Safe and Effective Gene Therapy

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Mrna Modification and Delivery Strategies Towards the Establishment of a Platform for Safe and Effective Gene Therapy Ghent University Faculty of Veterinary Medicine mRNA modification and delivery strategies towards the establishment of a platform for safe and effective gene therapy Oliwia Andries Dissertation submitted in fulfillment of the requirements for the degree of Doctor of Philosophy (PhD) 2015 Promotor: Co-Promotors: Prof. Dr. Niek Sanders Prof. Dr. Stefaan De Smedt Laboratory of Gene Therapy, Laboratory of Biochemistry and Department of Nutrition, Ethology and Physical Pharmacy, Genetics, Faculty of Pharmaceutical Sciences, Faculty of Veterinary Medicine, Ghent University Ghent University Dr. Tasuku Kitada Synthetic Biology Center Department of Biological Engineering, Massachusetts Institute of Technology 1 The author and the promoters give the authorization to consult and to copy parts of this thesis for personal use only. Any other use is limited by the Laws of Copyright, especially the obligation to refer to the source whenever results from this thesis are cited. De auteur en de promotoren geven de toelating dit proefschrift voor consultering beschikbaar te stellen en delen ervan te kopiëren voor persoonlijk gebruik. Elk ander gebruik valt onder beperkingen van het auteursrecht, in het bijzonder met betrekking tot de verplichting uitdrukkelijk de bron te vermelden bij het aanhalen van resultaten uit dit proefschrift. Ghent, March 25th 2015 Promotor: Author: Prof. dr. Apr. Niek N. Sanders Oliwia Andries, MSc Copromotors: Prof. dr. Apr. Stefaan De Smedt Dr. Tasuku Kitada 2 Here’s to the crazy ones. The rebels. The troublemakers. The ones who see things differently. While some may see them as the crazy ones, we see genius. Because the people who are crazy enough to think they can change the world, are the ones who do. - Apple Inc. 1997 3 Table of Contents LIST OF ABBREVIATIONS........................................................................................................ 9 CHAPTER 1............................................................................................................................ 13 GENERAL INTRODUCTION AND AIMS OF THE STUDY ..................................................... 13 GENERAL INTRODUCTION ................................................................................................... 14 AIMS OF THE STUDY ............................................................................................................. 16 I. LITERATURE REVIEW...................................................................................................... 17 CHAPTER 2 ............................................................................................................................ 18 MRNA AS A SAFE AND EFFECTIVE PLATFORM FOR GENE-BASED THERAPEUTICS ........................ 18 INTRODUCTION ...................................................................................................................... 19 Half-life and translatability of IVT mRNA ................................................................................. 19 Cap structure ............................................................................................................................ 19 Poly(A) tail ................................................................................................................................ 20 3’ and 5’ UTRs .......................................................................................................................... 21 mRNA platforms: modified and replicating .............................................................................. 22 The nuclear barrier: challenging for pDNA but irrelevant for mRNA ....................................... 25 Methods for mRNA delivery ..................................................................................................... 25 Electroporation and gene gun delivery..................................................................................... 27 Lipo- and polyplexes................................................................................................................. 28 Applications for mRNA as a drug molecule ............................................................................. 29 mRNA-loaded dendritic cells vaccine ...................................................................................... 29 Anti-cancer immunotherapy ..................................................................................................... 30 mRNA vaccine against infectious diseases ............................................................................. 31 Anti-allergy immunotherapy ..................................................................................................... 32 Passive immunoprophylaxis..................................................................................................... 32 Tailoring the immune responses for different applications ...................................................... 33 CONCLUSIONS ....................................................................................................................... 34 II. EXPERIMENTAL STUDIES .............................................................................................. 35 CHAPTER 3............................................................................................................................ 36 COMPARISON OF THE GENE TRANSFER EFFICIENCY OF MRNA/GL67 AND PDNA/GL67 COMPLEXES IN RESPIRATORY CELLS ......................................................................................... 36 INTRODUCTION ...................................................................................................................... 37 MATERIALS AND METHODS ................................................................................................. 39 Reagents .................................................................................................................................. 39 4 Plasmids ................................................................................................................................... 39 In vitro transcription of mRNA .................................................................................................. 40 Preparation and characterization of mRNA/GL67 and pDNA/GL67 complexes ..................... 40 In vitro transfection and protein expression measurements.................................................... 41 Mice and pulmonary delivery of the complexes....................................................................... 41 In Vivo Bioluminescence Imaging (BLI) ................................................................................... 42 Statistical Analysis.................................................................................................................... 42 RESULTS ................................................................................................................................. 42 Physicochemical characterization of mRNA/GL67 complexes prepared at different ratios ... 42 Transfection efficiency and cytotoxicity of the mRNA/GL67 complexes at different ratios in alveolar cells ............................................................................................................................. 44 Comparison of expression kinetics of mRNA/GL67 and pDNA/GL67 complexes .................. 46 Impact of cell division on transfection efficiency of mRNA/GL67 and pDNA/GL67 complexes .................................................................................................................................................. 47 The mRNA concentration during preparation of the complexes affects the transfection efficiency ................................................................................................................................... 49 In vivo transfection efficiency ................................................................................................... 50 Effect of serum on the physical properties and transfection efficacy of mRNA/GL67 and pDNA/GL67 complexes ............................................................................................................ 52 DISCUSSION ........................................................................................................................... 53 CONCLUSIONS ....................................................................................................................... 57 SUPPORTING INFORMATION AVAILABLE (APPENDIX B) ................................................. 58 CHAPTER 4............................................................................................................................ 59 INNATE IMMUNE RESPONSE AND PROGRAMMED CELL DEATH FOLLOWING CARRIER-MEDIATED DELIVERY OF UNMODIFIED MRNA TO RESPIRATORY CELLS........................................................ 59 INTRODUCTION ...................................................................................................................... 60 MATERIALS AND METHODS ................................................................................................. 61 Cell culture................................................................................................................................ 61 Plasmids ................................................................................................................................... 62 In vitro transcripton of mRNA ................................................................................................... 62 Preparation of complexes and transfection
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