Ecole Doctorale des Sciences de la Vie et de la Santé

Philippe Barthélemy

Description

Niveau: Supérieur, Doctorat, Bac+8
UNIVERSITÈ DE STRASBOURG Ecole Doctorale des Sciences de la Vie et de la Santé THÈSE présentée pour obtenir le grade de Docteur de l'Université de Strasbourg Discipline : Sciences du Vivant Domaine : Aspects Moléculaires et Cellulaires de la Biologie par Namrata JAIN Vecteurs de Gènes Non-Viraux Basés sur de Nouveaux Bolaamphiphiles Dissymétriques Soutenue le 21 décembre 2010 devant la commission d'examen : Pr. Philippe BARTHÉLÉMY Rapporteur externe Dr. Patrick MIDOUX Rapporteur externe Dr. Michel BESSODES Examinateur Dr. Guy ZUBER Rapporteur interne Dr. Andrey KLYMCHENKO Examinateur Dr. Guy DUPORTAIL Directeur de thèse UMR CNRS 7213, Faculté de Pharmacie, ILLKIRCH

• cellular studies


• significant transfection


• viral approach


• transfection efficiency


• vectors based


• ecole doctorale des sciences de la vie et de la santé

Sujets

• Louis Lebègue Duportail
• Delivery
• Richert
• Andrey
• Vauchelles
• Barthélemy
• Université de Strasbourg

Informations

• Publié par: profil-zyak-2012
• Publié le: 01 décembre 2010
• Nombre de lectures: 84
• Langue: Français
• Poids de l'ouvrage: 28 Mo

Extrait

UNIVERSITÈ DE STRASBOURG

Ecole Doctorale des Sciences de la Vie et de la Santé THÈSE

présentée pour obtenir le grade de Docteur de l’Université de Strasbourg Discipline : Sciences du Vivant Domaine : Aspects Moléculaires et Cellulaires de la Biologie par Namrata JAIN Vecteurs de Gènes Non-Viraux Basés sur de Nouveaux Bolaamphiphiles Dissymétriques

Soutenue le 21 décembre 2010 devant la commission d’examen :

Pr. Philippe BARTHÉLÉMY Dr. Patrick MIDOUX Dr. Michel BESSODES Dr. Guy ZUBER Dr. Andrey KLYMCHENKO Dr. Guy DUPORTAIL

Rapporteur externe Rapporteur externe Examinateur

Rapporteur interne

Examinateur

Directeur de thèse

UMR CNRS 7213, Faculté de Pharmacie, ILLKIRCH

UNIVERSITÈ DE STRASBOURG

Ecole Doctorale des Sciences de la Vie et de la Santé THÈSE

présentée pour obtenir le grade de Docteur de l’Université de Strasbourg Discipline : Sciences du Vivant Domaine : Aspects Moléculaires et Cellulaires de la Biologie par Namrata JAIN Non-Viral Gene Delivery Vectors Based on New Unsymmetrical Bolaamphiphiles

Soutenue le 21 décembre 2010 devant la commission d’examen :

Pr. Philippe BARTHÉLÉMY Dr. Patrick MIDOUX Dr. Michel BESSODES Dr. Guy ZUBER Dr. Andrey KLYMCHENKO Dr. Guy DUPORTAIL

Rapporteur externe Rapporteur externe Examinateur Rapporteur interne Examinateur Directeur de thèse

UMR CNRS 7213, Faculté de Pharmacie, ILLKIRCH

Acknowledgement My thesis project was performed in the Laboratoire de Biophotonique et Pharmacologie, directed by Prof. Yves MELY. I am grateful to him for providing me the opportunity to work in his lab. I would like to express my heartfelt gratitude to Director of my thesis Dr. Guy DUPORTAIL for his help, guidance and support during my research. He has been generous with his time and knowledge. I would like to express my deepest gratitude and sincere appreciation to my co-Director Dr. Andrey S. KLYMCHENKO for his supervision, inspiration and encouragement for monitoring my research. It has been a privilege to study with him. I am greatly honoured by the kind acceptance of Prof. Philippe BARTHÉLÉMY, Dr. Patrick MIDOUX, Dr. Michel BESSODES and Dr. Guy ZUBER as members of jury for my thesis and would like to thanks them for serving as my advisory committee. Special thanks to Dr. Valérie GOLDSCHMIDT for her guidance and support with cellular studies, and Dr. Youri ARNTZ for his help with AFM studies. I would like to extend my gratitude to Dr. Hugues de ROCQUIGNY for his help with HPLC, Dr. Sebnem ERCELEN for preliminary structural characterisation and cellular studies, Dr. Ludovic RICHERT and Dr. Romain VAUCHELLES for fluorescence imaging studies. I thank to the groups of Dr. J.-S. REMY and Dr. B. FRISCH for help with luminometry and DLS measurements. I would like to thank all my friends in the lab Kamal Kant SHARMA, Viktoriia POSTUPALENKO, Oleksandr KUCHERAK, Beata BASTA, Zeinab DARWICH, Julien GODET, Vanille GRENIER, Armelle JOUONANG, Jacques LUX and Hussein FTOUNI for their constant help, encouragement and our great discussions. I wish them all the best of luck in their future endeavours. I would also like to thank master’s students Nada KRAYEM and Ludovic JOUANOLOU with whom I worked. On a personal note, I would like to thank my family and friends who have supported me along the way in my pursuit for my goals.

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Abstract The potential of gene therapy to treat genetic disorders relies strongly on the efficiency of delivery vectors. The failure of viral gene therapy clinical trails due to toxicity, immunogenicity and carcinogenicity raised serious safety concerns and strongly motivated a non-viral approach. However, non-viral gene delivery, mainly based on lipids and polymers, is currently ineffective forin vivoapplications due to poor structural control of their DNA complexes and lack of serum stability. The research conducted for this thesis was aimed at the design and synthesis of unsymmetrical bolaamphiphiles as a new family of non-viral vectors. For this purpose, several new bolaamphiphiles (bolas) were obtained by multi-step organic synthesis, bearing neutral sugar residue (mannoic, gluconic, lactonic acid or PEG group) and mono- or oligo-cationic ammonium-based headgroups, connected by different hydrophobic spacers. Within this design, a positively charged headgroup is expected to bind DNA, the hydrophobic spacer should drive the formation of a monolayer membrane shell around DNA, while the neutral group is to be exposed outside that will prevent further aggregation of the complexes. Our different structural characterization measurements showed that the self-assembly of bola, their interaction with DNA and the morphology of the bolaplexes depend strongly on the bola structure. While the first two generations of bolas lack the essential features of nonviral vectors, the final third generation was found highly promising. The latter showed strong interaction with DNA and formation of small DNA complexes (~100 nm) at low N/P, while at higher N/Ps it was significantly larger due to neutralization of their surface charge. Atomic Force Microscopy studies revealed a rod shaped or spherical nano-structural morphology of the bolaplexes. Their transfection efficiency and intracellular trafficking were tested in different cell lines. Significant transfection efficiency was observed in the presence of helping agents like DOPE or chloroquine suggesting that the key barrier for their internalization is the endosomal escape. Some bolaplex formulations showed transfection efficiency comparable to the best commercial transfection agents. Finally, all bolas showed low cytotoxicity. Thus, the present work validates a new concept for construction of nonviral vectors featuring controlled small size, high efficiency and low cytotoxicity. We believe that the bola structure could be further modified to enhance transfection efficiency by favouring endosomal escape without use of any helping agent.

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Table of Contents

Contents

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Contents ABBREVIATIONS................................................................ 7 GENERAL INTRODUCTION ................................................ 9 BIBLIOGRAPHIC REVIEW ................................................ 11 1.GENE THERAPY .................................................................... 12 1.1Overview and Historical Prospective....................................... 12 1.2.................. 14In Vitro (or Ex Vivo) and In Vivo Gene Therapy 2.15GENE DELIVERY BARRIERS ............................................... 2.1............................................................... 16Extracellular Barriers 2.1.1..................................................................Specific targeting 17 2.2Intracellular Barriers ................................................................ 20 2.2.1.................................Cellular uptake and endosomal escape 20 2.2.2Cytosolic mobility and nuclear entry....................................22 3.GENE DELIVERY VECTORS................................................. 23 3.1Viral Vectors .............................................................................. 23 3.1.1Retrovirus (including lentivirus) ..........................................24 3.1.2Adenovirus............................................................................26 3.1.3Adeno-Associated viruses ....................................................27 3.1.4Herpes simplex virus ............................................................27 3.2Non-Viral Vectors...................................................................... 28 3.2.1Naked DNA ..........................................................................29 3.2.2Electroporation .....................................................................30 3.2.3...............................................................................Gene gun 31 3.2.4Cationic lipid based vectors..................................................32 3.2.5..........................................................Polymer based vectors 40 4...... 47Intracellular Trafficking of Lipoplexes and Polyplexes 4.1Cellular Binding and Uptake.................................................... 47 4.2..................................................................... 49Endosomal Escape 4.2.1Lipoplexes.............................................................................49 4.2.2Polyplexes .............................................................................51 4.3Nuclear Entry............................................................................. 53 4.3.1Lipoplexes.............................................................................53 4.3.2Polyplexes .............................................................................54

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Contents 5.BOLAAMPHIPHILE-BASED VECTORS................................ 55 5.1General Introduction................................................................. 55 5.2........................................... 58Self-Assembly of Bolaamphiphiles 5.3Effect of Hydrophobic Spacer Chain Length and Headgroup…….... ................................................................................ 60 5.4Bolas as Gene Delivery Vectors................................................ 62 6.IDEAL VECTOR ..................................................................... 64 MATERIALS AND METHODS ........................................... 65 1.Synthesis of Bolaamphiphiles ............................................. 66 1.1First Generation Bolas .............................................................. 66 1.2Second Generation Bolas .......................................................... 76 1.3Third Generation Bolas ............................................................ 81 1.4Synthesis of Some Intermediates.............................................. 92 2.95Fluorescent Probes............................................................... 2.1..................................................................... 95Ethidium Bromide 2.2 1,8-ANS ...................................................................................... 96 2.396YOYO-1 ...................................................................................... 3.Plasmids ................................................................................ 97 3.1Amplification and Purification................................................. 97 3.2Determination of the Plasmid Concentration ......................... 98 4.98Preparation of Vector/DNA complexes ............................... 4.1DNA Complexes with Bolas (bolaplexes) ................................ 98 4.2DNA Complexes with Bola:DOPE (1:1) .................................. 99 4.3DNA Complex with jetPEI ....................................................... 99 5.100Physical Measurements ..................................................... 5.1........................................................ 100Absorption Spectroscopy 5.2Steady-State Fluorescence Spectroscopy............................... 100 5.3Dynamic Light Scattering Measurements............................. 101 5.4Zeta Potential Measurements ................................................. 103 5.5Confocal Laser Scanning Microscopy ................................... 105 5.6Atomic Force Microscopy (AFM) .......................................... 105 6.............................................. 106Agarose Gel Electrophoresis

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Contents 7.Cell Culture and Transfection ............................................ 107 7.1General Conditions and Cell Lines ........................................ 107 7.2Luciferase Assay ...................................................................... 108 7.3Protein Assay............................................................................ 109 7.4MTT Assay (Cell Viability)..................................................... 110 RESEARCH AIM .............................................................. 111 RESULTS AND DISCUSSION ......................................... 114 1.First Generation Bolas: bearing mono-cationic and sugar headgroups ................................................................................ 115 2.Second Generation Bolas: bearing di-cationic and sugar headgroups ................................................................................ 127 3.Third Generation Bolas: bearing di-cationic & sugar/PEG headgroups and varied hydrophobic spacer.......................... 134 3.1Bolas with Gluconic Headgroup............................................. 140 New unsymmetrical bolaamphiphiles: synthesis, assembly with DNA and application for gene delivery (Publication 1) ............................143 3.2............................................. 153Bolas with Lactonic Headgroup Lactose-ornithine bolaamphiphiles for efficient gene delivery in vitro (Publication 2)…… ..........................................................................146 3.3Bolas with PEG Headgroup.................................................... 149 GENERAL CONCLUSIONS AND FUTURE PROSPECTIVES .............................................................. 149 REFERENCES.................................................................. 158 APPENDIX ........................................................................ 160 Excited-State Intramolecular Proton Transfer Distinguishes Microenvironments in Single-And Double-Stranded DNA (Publication 3)........................................................................................ 162 Virus-sized DNA nanoparticles for gene delivery based on micelles of cationic calixarenes (Publication 4) ..................................................... 184

Résumé en Français………………………………………..184 PUBLICATIONS ............................................................... 185

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ABBREVIATIONS

AAV AFM 1,8-ANS ASGPR BC BOP

bp CAR Cbz Ch  CMC CT-DNA DIEA DLS DMEM DMF DMSO DOPC DOPE EtBr EM FBS HCl HIV HOBt HSV kb kbp Luc

Adeno-associated virus Atomic Force Microscope 1-Anilinonaphthalene-8-Sulfonic Acid Asialoglycoprotein receptors Bicinchoninic Acid

Abbreviations

(Benzotriazol-1-yloxy)tris(dimethylamino)phosphonium hexafluorophosphate Base pair Coxsackie and Adenovirus Receptor Benzyloxy Carbonyl Cholesterol Critical micelle concentration Calf Thymus DNA N,N-Diisopropylethylamine Dynamic Light Scattering Dulbecco’s modified eagle’s medium Dimethyl formamide Dimethyl sulfoxide Dioleoylphosphatidylcholine Dioleoyl phosphatidylethanolamine Ethidium bromide Electron Microscopy Fetal bovine serum Hydrochloric acid Human Immunodeficiency Virus 1-hydroxybenzotriazole Herpes simplex virus Kilo base Kilo base pair Luciferase

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LL LUV LC-MS MES

MLM MS MTT NLS NMR Opti-MEM PAM PBS pCMV-Luc

pDNA Pd Pd-C PE PEG PEI PLL RLU RME RT TAE TFA TfR THF YOYO-1

Abbreviations

Lactonolactone Large unilamellar vesicles Liquid chromatography-mass spectrometry 2-(N-morpholino)-ethane sulphonic acid Monolayer membrane Mass Spectrometry 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyltetrazolium bromide

Nuclear localization signal Nuclear Magnetic Resonance Reduced Serum Media Polyamidoamine Phosphate buffer saline Plasmid encoding the photinus pyralis luciferase gene under the control of the cytomegalovirus promoter Plasmid DNA Palladium Palladium on carbon Posphatidylethanolamine Polyethylene glycol Polyethylenimine Poly(l-lysine) Relative light units Receptor-mediated endocytosis Room Temperature Tris-acetate-ethylenediaminetetraacetic acid buffer Trifluoroacetic acid Transferrin receptor Tetrahydrofuran 1,1’-(4,4,8,8-tetramethyl-4,8 diazaundecamethylene)bis[4-[[3- methylbenz-1,3-oxazol-2-yl]methylidine]-1,4 dihydroquinolinium] tetraiodide

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