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Enhancement of Virus-Like Particle Production in Hek293 Cell Cultures

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Enhancement of Virus-Like Particle Production in Hek293 Cell Cultures ADVERTIMENT. Lʼaccés als continguts dʼaquesta tesi queda condicionat a lʼacceptació de les condicions dʼús establertes per la següent llicència Creative Commons: http://cat.creativecommons.org/?page_id=184 ADVERTENCIA. El acceso a los contenidos de esta tesis queda condicionado a la aceptación de las condiciones de uso establecidas por la siguiente licencia Creative Commons: http://es.creativecommons.org/blog/licencias/ WARNING. The access to the contents of this doctoral thesis it is limited to the acceptance of the use conditions set by the following Creative Commons license: https://creativecommons.org/licenses/?lang=en 66 UNIVERSITAT AUTÒNOMA DE BARCELONA ESCOLA D’ENGINYERIA DEPARTAMENT D’ENGINYERIA QUÍMICA, BIOLÒGICA I AMBIENTAL PROGRAMA DE DOCTORAT EN BIOTECNOLOGIA ENHANCEMENT OF VIRUS-LIKE PARTICLE PRODUCTION IN HEK293 CELL CULTURES JAVIER FUENMAYOR GARCÉS PHD THESIS APRIL 2018 Advisors: Dr. Francesc Gòdia Dr. Laura Cervera UNIVERSITAT AUTÒNOMA DE BARCELONA ESCOLA D’ENGINYERIA DEPARTAMENT D’ENGINYERIA QUÍMICA, BIOLÒGICA I AMBIENTAL PROGRAMA DE DOCTORAT EN BIOTECNOLOGIA ENHANCEMENT OF VIRUS-LIKE PARTICLE PRODUCTION IN HEK293 CELL CULTURES PhD thesis presented by Javier Fuenmayor Garcés to apply for the degree of Doctor in Biotechnology by Universitat Autònoma de Barcelona. This work has been performed at Departament d’Enginyeria Química, Biològica i Ambiental from Universitat Autònoma de Barcelona with the supervision of Dr. Francesc Gòdia and Dr. Laura Cervera. Bellaterra, April 2018. Dr. Francesc Gòdia Dr. Laura Cervera Table of contents TABLE OF CONTENTS ABBREVIATIONS ................................................................................................... 7 RESUM .................................................................................................................. 9 SUMMARY .......................................................................................................... 13 INTRODUCTION .................................................................................................. 17 1. Viral vaccines ............................................................................................................... 19 1.1. Types of vaccines ................................................................................................................. 19 1.2. Types of virus-like particles .................................................................................................. 20 2. Human immunodeficiency virus (HIV) .......................................................................... 21 2.1. HIV structure and infective cycle ......................................................................................... 21 2.2. HIV treatment and vaccination ............................................................................................ 22 2.3. HIV VLPs ............................................................................................................................... 23 3. Production platform for VLP production ...................................................................... 24 3.1. Bacteria and yeast ................................................................................................................ 25 3.2. Baculovirus/Insect Cell (B/IC) expression system ................................................................ 26 3.3. Plants .................................................................................................................................... 27 3.4. Mammalian cells .................................................................................................................. 27 3.5. Production yields.................................................................................................................. 28 4. VLP production approaches in mammalian cell platforms ............................................ 29 4.1. Transient gene expression ................................................................................................... 30 4.2. Methods for enhancing transient transfection .................................................................... 32 4.2.1. Origins of replication .................................................................................................... 32 4.2.2. PEI:DNA concentration optimization ............................................................................ 33 4.2.3. Extended gene expression ............................................................................................ 33 4.2.4. Chemical additives ........................................................................................................ 34 4.2.5. Gene inhibitors ............................................................................................................. 35 4.3. Stable gene expression ........................................................................................................ 35 4.4. TGE at bioreactor scale ........................................................................................................ 37 5. References .................................................................................................................. 40 OBJECTIVES ......................................................................................................... 53 RESULTS .............................................................................................................. 57 1. ENHANCEMENT OF HIV-1 VLP PRODUCTION USING GENE INHIBITION STRATEGIES .......... 59 2. EXTENDED GENE EXPRESSION FOR GAG VLP PRODUCTION ACHIEVED AT BIOREACTOR SCALE......................................................................................................... 93 5 Table of contents 3. TRANSIENT GENE EXPRESSION OPTIMIZATION AND EXPRESSION VECTOR COMPARISON TO IMPROVE HIV-1 VLP PRODUCTION IN HEK293 CELL LINES .................. 123 4. STABLE CELL LINE GENERATION FOR THE PRODUCTION OF GAGGFP VLPS .................. 155 DISCUSSION AND FUTURE DIRECTIONS ............................................................. 187 CONCLUSIONS ................................................................................................... 209 6 Abbrebiations ABBREVIATIONS AAV Adeno-associated vector AAVS1 Adeno-associated virus site 1 ADD Additives AIDS Acquired immune deficiency syndrome ANOVA Analysis of variance ART Antiretroviral therapy ATF Alternating tangential flow B/IC Baculovirus-insect cell CCD Central composite design CHO Chinese hamster ovary cells CMV Citomegalovirus CRISPR Clustered regulatory interspaced short palindromic repeats dpt Day post transfection EBNA-1 Epstein-Barr virus nuclear antigen 1 EBV Epstein-Barr virus EGE Extended gene expression ELISA Enzyme-linked immunosorbent assay Env Envelope FACS Fluorescence activated cell sorting FITC Fluorescein isotiocyanate FU Fluorescence unit GFP Green fluorescence protein GOI Gene of interest gp120 Glycoprotein 120 gp410 Glycoprotein 41 HA Hemagglutinin HCD Host cell DNA HCP Host cell protein HDAC Histone deacetylase HEK293 Human embryonic kidney cells HF Hollow fiber HIV Human immunodeficiency virus hpt Hour post transfection HPV Human papilloma virus IRNA interference RNA M1 Matrix protein 1 mAb Monoclonal antibody 7 Abbrebiations ME Medium exchange NIH National Institute of Health NTA Nanoparticle tracking analysis OriP Origen of replication P p24 Protein 24 (CA subunit from HIV-1 Gag polyprotein) PCR Polymerase chain reaction PDE Phosphodiesterase PEI Polyethyleneimine PI Propidium iodide PPP1R12C Phosphatase 1 regulatory subunit 12C PTM Post-translational modification qPCR Quantitative polymerase chain reaction RFU Relative fluorescence unit RISC RNA-induced silencing complex rpm Revolutions per minute SD Standard deviation SGE Stable gene expression shRNA Short harping RNA SIV Simian immunodeficiency virus SV40 Simian vacuolating virus 40 SV40ori Simian vacuolating virus 40 origen of replication TALENS Transcription activator-like effector nucleases TFF Tangential flow filtration TGE Transient gene expression UC Ultracentrifugation VLP Virus-like particle VVD Volume of feed per bioreactor volume per day ZFN Zinc finger nuclease λem Emission wavelength (nm) λex Excitation wavelength (nm) 8 Resum Resum Les virus-like particles (VLP) tenen un gran potencial com a candidats pel desenvolupament de vacunes. En aquest treball, s’han aplicat diverses estratègies per la millora de la producció de VLPs (basades en la poliproteïna Gag del VIH-1) en HEK293 mitjançant la transfecció transitòria i l’expressió estable. Aquest candidat té aplicacions potencials en el desenvolupament d’una vacuna terapèutica contra la SIDA. Tres capítols del treball descriuen estratègies basades en la transfecció transitòria, mentre que l’últim capítol consisteix en la generació d’una línea HEK293 estable per la producció de VLPs de VIH-1. En el primer capítol, es va dur a terme la combinació de l’estratègia extended gene expression (EGE) amb la complementació del medi amb additius químics. L’EGE (basada en la repetició de diversos recanvis de medi i retransfeccions) ha reportat una millora en la producció de VLPs de 12 cops mentre que la complementació química del medi amb potenciadors de l’expressió gènica comporta una millora de 4 cops. La combinació de l’EGE amb additius químics va resultar en una millora de 1.5 cops en la producció de VLPs de VIH- 1 comparat amb l’EGE sola. Com alternativa a l’ús d’additius químics, es va provar l’expressió de shRNA que produeixin el mateix efecte sobre les cèl·lules. Aquesta estratègia innovadora
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