Ecole Doctorale des Sciences de la Vie et de la Santé

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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


Publié le : mercredi 1 décembre 2010
Lecture(s) : 110
Source : scd-theses.u-strasbg.fr
Nombre de pages : 222
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UNIVERSITÈ DE STRASBOURG
Ecole Doctorale des Sciences de la Vie et de la SantéTHÈSE
présentée pour obtenir le grade deDocteur de l’Université de StrasbourgDiscipline : Sciences du VivantDomaine : 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 externeRapporteur 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 SantéTHÈSE
présentée pour obtenir le grade deDocteur de l’Université de StrasbourgDiscipline : Sciences du VivantDomaine : 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 externeRapporteur externe Examinateur Rapporteur interne ExaminateurDirecteur 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 ................................................ 9BIBLIOGRAPHIC REVIEW ................................................ 111.GENE THERAPY .................................................................... 121.1Overview and Historical Prospective....................................... 121.2.................. 14In Vitro (or Ex Vivo) and In Vivo Gene Therapy 2.15GENE DELIVERY BARRIERS ............................................... 2.1............................................................... 16Extracellular Barriers 2.1.1..................................................................Specific targeting 172.2Intracellular Barriers ................................................................ 202.2.1.................................Cellular uptake and endosomal escape 202.2.2Cytosolic mobility and nuclear entry....................................223.GENE DELIVERY VECTORS................................................. 233.1Viral Vectors .............................................................................. 233.1.1Retrovirus (including lentivirus) ..........................................243.1.2Adenovirus............................................................................263.1.3Adeno-Associated viruses ....................................................273.1.4Herpes simplex virus ............................................................273.2Non-Viral Vectors...................................................................... 283.2.1Naked DNA ..........................................................................293.2.2Electroporation .....................................................................303.2.3...............................................................................Gene gun 313.2.4Cationic lipid based vectors..................................................323.2.5..........................................................Polymer based vectors 404...... 47Intracellular Trafficking of Lipoplexes and Polyplexes 4.1Cellular Binding and Uptake.................................................... 474.2..................................................................... 49Endosomal Escape 4.2.1Lipoplexes.............................................................................494.2.2Polyplexes .............................................................................514.3Nuclear Entry............................................................................. 534.3.1Lipoplexes.............................................................................534.3.2Polyplexes .............................................................................54
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Contents 5.BOLAAMPHIPHILE-BASED VECTORS................................ 555.1General Introduction................................................................. 555.2........................................... 58Self-Assembly of Bolaamphiphiles 5.3Effect of Hydrophobic Spacer Chain Length and Headgroup…….... ................................................................................ 605.4Bolas as Gene Delivery Vectors................................................ 626.IDEAL VECTOR ..................................................................... 64MATERIALS AND METHODS ........................................... 651.Synthesis of Bolaamphiphiles ............................................. 661.1First Generation Bolas .............................................................. 661.2Second Generation Bolas .......................................................... 761.3Third Generation Bolas ............................................................ 811.4Synthesis of Some Intermediates.............................................. 922.95Fluorescent Probes............................................................... 2.1..................................................................... 95Ethidium Bromide 2.2 1,8-ANS ...................................................................................... 962.396YOYO-1 ...................................................................................... 3.Plasmids ................................................................................ 973.1Amplification and Purification................................................. 973.2Determination of the Plasmid Concentration ......................... 984.98Preparation of Vector/DNA complexes ............................... 4.1DNA Complexes with Bolas (bolaplexes) ................................ 984.2DNA Complexes with Bola:DOPE (1:1) .................................. 994.3DNA Complex with jetPEI ....................................................... 995.100Physical Measurements ..................................................... 5.1........................................................ 100Absorption Spectroscopy 5.2Steady-State Fluorescence Spectroscopy............................... 1005.3Dynamic Light Scattering Measurements............................. 1015.4Zeta Potential Measurements ................................................. 1035.5Confocal Laser Scanning Microscopy ................................... 1055.6Atomic Force Microscopy (AFM) .......................................... 1056.............................................. 106Agarose Gel Electrophoresis
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Contents 7.Cell Culture and Transfection ............................................ 1077.1General Conditions and Cell Lines ........................................ 1077.2Luciferase Assay ...................................................................... 1087.3Protein Assay............................................................................ 1097.4MTT Assay (Cell Viability)..................................................... 110RESEARCH AIM .............................................................. 111RESULTS AND DISCUSSION ......................................... 1141.First Generation Bolas: bearing mono-cationic and sugar headgroups ................................................................................ 1152.Second Generation Bolas: bearing di-cationic and sugar headgroups ................................................................................ 1273.Third Generation Bolas: bearing di-cationic & sugar/PEG headgroups and varied hydrophobic spacer.......................... 1343.1Bolas with Gluconic Headgroup............................................. 140New unsymmetrical bolaamphiphiles: synthesis, assembly with DNA and application for gene delivery (Publication 1) ............................1433.2............................................. 153Bolas with Lactonic Headgroup Lactose-ornithine bolaamphiphiles for efficient gene delivery in vitro (Publication 2)…… ..........................................................................1463.3Bolas with PEG Headgroup.................................................... 149GENERAL CONCLUSIONS AND FUTURE PROSPECTIVES .............................................................. 149REFERENCES.................................................................. 158APPENDIX ........................................................................ 160Excited-State Intramolecular Proton Transfer Distinguishes Microenvironments in Single-And Double-Stranded DNA (Publication 3)........................................................................................ 162Virus-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 DMSODOPC DOPEEtBr 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 DNAN,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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General Introduction
GENERAL INTRODUCTION Gene therapy is a rapidly developing area of research representing a fundamentally new way to treat a disease. Genetic material (genes) used in such a therapy should express a protein or interfere with its synthesis inside the cell. Replacing a defective gene with a normal gene and thus restoring the lost gene function in the patient’s body is the essence of gene therapy.
The success in gene therapy strongly relies on the efficiency of delivery vectors. An ideal vector should be able to protect gene from all cellular barriers for effective delivery to the nucleus, should have the ability to regulate expression of the gene of interest and minimize toxicity by targeting specific cells. A variety of gene delivery vectors, viral and non-viral vectors, have been developed in past decades. Although viral vectors are highly efficient in transfecting cells, there are several concerns that limit their use to deliver DNA therapeutics in humans. The major concern is the toxicity of the viruses and their potential for generating a strong immune response. All these concerns make non-viral vectors, based on lipids and polymers, an attractive alternative. Their key advantages over viral vectors are: simplicity of use, large-scale production, ability to carry DNAs of large size, and lack of immune response. Their complexes with DNA (lipoplexes and polyplexes, respectively) show high efficiencyin vitro, though their application for gene therapyin vivoa challenge. One of the key problems with these vectors is the remains poor structural control of their complexes with DNA. The vast majority of natural and synthetic lipids studied so far are “monopolar” amphiphiles, i.e. molecules presenting one polar headgroup and generally two hydrophobic chains. However, bipolar amphiphiles, which are analogues of lipids presenting polar groups at the two opposite sides of the hydrophobic chain(s), so-called bolaamphiphiles (bolas), became the matter of intensive research only in the recent years. A remarkable feature of bolas is that, in contrast to bilayer membranes formed by lipids, they can form monolayer membranes due to the presence of membrane-spanning hydrophobic chains responsible for enhanced physical stability of bola membranes. In nature, they are mainly present in archaebacteria and ensure the integrity of the bacterial membrane at critical temperature and pH. In this context, unsymmetrical bolas, bearing
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