Improving Biopharmaceutical Production of Chinese Hamster Ovary Cells using Targeted Genome Engineering Tools A thesis submitted for the degree of PhD Dublin City University By Kevin Kellner, M.Sc. The research work described in this thesis was conducted under supervision of Dr. Nga Lao and Prof. Martin Clynes Dr. Niall Barron, University College Dublin National Institute for Cellular Biotechnology School of Biotechnology Dublin City University October 2018 ___________________________________________________________________________ I hereby certify that this material, which I now submit for assessment on the programme of study leading to the award of Ph.D. is entirely my own work, that I have exercised reasonable care to ensure that the work is original, and does not to the best of my knowledge breach any law of copyright, and has not been taken from the work of others save and to the extent that such work has been cited and acknowledged within the text of my work. Signed: ____________ (Candidate) ID No.: 14212905 Date: _______ ___________________________________________________________________________ I ___________________________________________________________________________ Dedicated to my beloved parents and friends ___________________________________________________________________________ II ___________________________________________________________________________ Acknowledgements Almost 5 years ago when I moved to Ireland to write my master’s thesis I would have never thought that I will stay a few years longer to do my PhD as well. With that I’d like to express my sincere gratitude to Dr. Niall Barron who gave me the chance to do my PhD in his lab. Thanks for never loosing your patience in every situation and for being always encouraging and positive even when experiments didn’t go well. On the same hand I’d like to say thanks to Prof. Martin Clynes who is the most supportive and good person I’ve ever met! Thanks also to Dr Nga Lao for supervising the last year of my thesis as well as managing that bunch of students in the lab - what would we do without your magic cloning hands? I would also like to thank the whole proteomics team with Dr. Paula Meleady, Dr. Micheal Henry, Orla Coleman, Dr. Shane Kelly and Dr. Mark Gallagher for helping and contributing so much towards my work. Thanks also to Dr. Clair Gallagher for spending a lot of hours on the FACS – still haven’t figured out your secret of keeping the patience! Of course, I would also like to thank all the postgrads and graduated doctors in our institute and School of Biotechnology for great lunch breaks, walks to Spar/Londis and coffee breaks. I am very glad to have made very good friends during my time in Ireland! Thanks Orla, Laura, Niamh, Giuseppe, Paul, Gemma, Andrew and Alan for the great company and laughs over the last few years. You have all been an amazing support over the last years especially in times of the reoccurring “PhD slumps”. Special thanks also to Justine Meiller, Mairead Callen, Emer Walsh, Gillian Smith and Carol McNamara for your support and help over the last years. The place would not run without you! Especially I would like to express my sincere gratitude to David Willaert for being the most amazing mentor and manager. I feel like I learned more over the last year as part of your team than during my entire PhD. Besides that, my last year wouldn’t have gone so smoothly without the motivating and inspiring speaches every second Friday. Thanks for always believing in me! I also want to thank everyone in the academia sales team for being outstanding and amazing - It was a pleasure to work with each of you and I am glad that I can call all of you friends by now. I would also like to express my deepest gratitude and appreciation to my best friends: Evelyn, Mischa, Caio. Nora and Maggy. Even though we did not see each other as often as I would have liked to during my time in Ireland I am deeply grateful that you have all been there for me at any time and that I could always count on you! Thanks also for some awesome visits and travels through Ireland and the very best support during times of discouragement but also excitement. There are no words which could express my gratitude to my beloved parents Angelika and Erwin for always supporting me at every stage during my life and not only throughout my PhD. Thanks for always having my back, inspring me to follow my dreams but also for pushing me always a little into the right direction! I could not have done it without you. ___________________________________________________________________________ III ___________________________________________________________________________ Scientific Talks, Posters, Publications and Graduate Training Elements Graduate Training Elements: - Leadership in Research - Biopharma Industry Regulations and Management - Posgraduate Tuter and Demonstrator - Professional Skills for Scientists Selected Scientific Talks: Cell Line Development and Engineering US 12-14th June 2018 in San Francisco as invited speaker. 28th European Society for Animal Cell Culture Technology UK (ESACT-UK) 10-11th January 2018 in Leeds, Poster Flash Presentation BioProduction Congress 2017 8-10th October 2017 in Dublin, Poster Flash Presentation Publications: Kellner K., Solanki A., Amann T., Lao N. and Barron N. (2018): "Targeting miRNAs with CRISPR/Cas9 to Improve Recombinant Protein Production of CHO Cells"; Methods Molecular Biology (David L. Hacker), 1850, - accepted for publication. Kellner K., Lao N., Coleman O., Meleady P. and Barron N. (2018): “Molecular impact of the microRNA-23 cluster on bioprocess relevant attributes of Chinese hamster ovary cells”; BMC Proceedings 2018 (12 - Suppl 1). ___________________________________________________________________________ IV ___________________________________________________________________________ Table of contents 1 Introduction ........................................................................................................................ 3 1.1 CHO cells in biopharmaceutical industry .............................................................. 3 1.1.1 Cell culture regimes and formats .................................................................... 5 1.1.2 CHO cell heterogeneity..................................................................................... 9 1.2 CHO cell engineering ............................................................................................. 11 1.2.1 Implication of ‘omic approaches .................................................................... 11 1.2.2 Apoptosis engineering .................................................................................... 12 1.2.3 Metabolic engineering ..................................................................................... 13 1.2.4 Secretion and molecular chaperone engineering ....................................... 14 1.2.5 Promoter engineering and optimisation of expression systems .............. 15 1.2.6 Glycosylation-engineering .............................................................................. 16 1.3 MicroRNAs ............................................................................................................... 18 1.3.1 Biogenesis of miRNAs .................................................................................... 20 1.3.2 MicroRNAs as potential tools for cell line engineering .............................. 24 1.3.3 Biological function of the miR-23-24-27 cluster and its role in disease .. 25 1.4 Targeted genome engineering tools .................................................................... 29 1.4.1 Sponge decoy technology for the stable or transient depletion of miRNAs 29 1.4.2 Clustered regularly interspaced short palindromic repeats (CRISPR) .... 31 1.4.3 CRISPR/Cas9 as targeted genome engineering tool ................................ 35 2 Aims of the project ......................................................................................................... 41 3 Material and Methods .................................................................................................... 43 ___________________________________________________________________________ V ___________________________________________________________________________ 3.1 Cell culture techniques ........................................................................................... 43 3.1.1 Ultrapure water................................................................................................. 43 3.1.2 Glassware ......................................................................................................... 43 3.1.3 Cell culture cabinet .......................................................................................... 43 3.1.4 Culturing of mammalian cells ......................................................................... 44 3.1.5 Cell lines and expressed products ................................................................ 48 3.1.6 Cryopreservation of cells ................................................................................ 48 3.1.7 Thawing of cryopreserved cells ..................................................................... 48 3.1.8 Lipid- and PEI-based transfection methods ................................................ 49 3.1.9 Physical transfection methods – Electroporation ....................................... 50 3.1.10 Generation of lentiviral particles ................................................................ 51 3.1.11 Limited