Cell-surface receptor specific delivery of short interfering RNAs via bivalent aptamer-siRNA transcripts or covalent antibody-siRNA conjugates [Elektronische Ressource] / vorgelegt von Ulrich Wüllner

Ulli Weigel

Sujets

• Biologie

Informations

• Publié par: rheinisch-westfalischen_technischen_hochschule_-rwth-_aachen
• Publié le: 01 janvier 2007
• Nombre de lectures: 4
• Langue: English
• Poids de l'ouvrage: 4 Mo

Extrait

Cell-surface receptor specific delivery of
short interfering RNAs via
bivalent aptamer-siRNA transcripts or
covalent antibody-siRNA conjugates


Von der Fakultät für Mathematik, Informatik und Naturwissenschaften
der
Rheinisch-Westfälischen Technischen Hochschule Aachen
zur Erlangung des akademischen Grades eines
Doktors der Naturwissenschaften genehmigte Dissertation
vorgelegt von

Diplom-Chemiker
Ulrich Wüllner
aus Bielefeld


Berichter: Universitätsprofessor Dr. rer. nat. Rainer Fischer
Universitätsprofessor Dr. rer. nat. Dr. rer. medic. Stefan Barth

Tag der mündlichen Prüfung: 12. Dezember 2007

Diese Dissertation ist auf den Internetseiten der Hochschulbibliothek online
verfügbar Content

1 Introduction............................................................................................... 7
1.1 RNA interference.............................................................................................. 7
1.2 RNA interference for cancer therapy................................................................ 9
1.3 Aptamers......................................................................................................... 11
1.4 Eucaryotic elongation factor 2........................................................................ 13
1.5 Delivery strategies for RNA interference based drug..................................... 14
1.6 Prostate specific membrane antigen (PSMA)................................................. 18
1.7 The lymphocyte activation marker CD30....................................................... 19
1.8 Aim of the thesis............................................................................................. 19
2 Materials and Methods ........................................................................... 22
2.1 Materials ......................................................................................................... 22
2.1.1 Chemicals ......................................................................................................................... 22
2.1.2 Media stock solutions and buffers .................................................................................... 22
2.1.3 Standard buffer and media compositions.......................................................................... 22
2.1.4 Buffers for RNA work ...................................................................................................... 24
2.1.5 Antibodies 25
2.1.6 Reaction kits and Enzymes ............................................................................................... 26
2.1.7 Synthetic oligonucleotides................................................................................................ 26
2.1.8 Synthetic siRNA sequences and targets............................................................................ 28
2.1.9 RNA-Aptamer sequences ................................................................................................. 30
2.1.10 Bacterial strains and Vector Systems................................................................................ 31
2.1.11 Equipment and applications.............................................................................................. 32
2.2 Methods .......................................................................................................... 33
2.2.1 Cell culture........................................................................................................................ 33
2.2.2 Electrophoresis protocols.................................................................................................. 33
2.2.3 Methods for the evaluation of siRNA mediated gene silencing........................................ 34
2.2.4 Methods for the preparation of RNA aptamers................................................................. 36 4 Content
2.2.5 Aptamer-siRNA transcript evaluation............................................................................... 40
2.2.6 Antibody-siRNA conjugate preparation............................................................................ 42
2.2.7 Antibody siRNA conjugate characterization..................................................................... 45
3 Results ..................................................................................................... 46
3.1 Characterization of siRNA targeting eucaryotic elongation factor 2 (EEF2) .46
3.1.1 Transfection efficiency 46
3.1.2 Establishment of a q-PCR protocol for EEF2 mRNA expression..................................... 47
3.1.3 Gene silencing efficiency of siEEF2 sequence 1 and 2..................................................... 50
3.1.4 Evaluation of EEF2 knock down on protein level ............................................................ 51
3.1.5 Evaluation of the cytotoxicity of siEEF2 .......................................................................... 52
3.1.6 Summary........................................................................................................................... 53
3.2 Evaluation of aptamer siRNA transcripts........................................................54
3.2.1 Design of bivalent aptamer siRNA transcripts.................................................................. 54
3.2.2 Assembly PCR of bivalent aptamer siRNA DNA templates ............................................ 57
3.2.3 In vitro transcription of bivalent aptamer siRNA transcripts 59
3.2.4 RNA Fluoresceine labeling............................................................................................... 60
3.2.5 Analysis of binding specificity of bivalent aptamer siRNA transcripts ............................ 61
3.2.6 Functional affinity measurements ..................................................................................... 62
3.2.7 Internalization assays ........................................................................................................ 64
3.2.8 In vitro DICER assays....................................................................................................... 67
3.2.9 Evaluation of the cytotoxic potential of the bivalent constructs x-PSM-B1-siEEF2 and x-
PSM-B2-siEEF2 ............................................................................................................... 68
3.2.10 Interferon β detection assay .............................................................................................. 72
3.2.11 Quantitative cell viability assays....................................................................................... 73
3.2.12 Modular annealing bivalent aptamer-siRNA transcripts................................................... 74
3.2.13 Summary........................................................................................................................... 77
3.3 Evaluation of siRNA-antibody conjugation strategies....................................78
3.3.1 Conjugation via reductive amination ................................................................................ 78
3.3.2 Conjugation via N-(3-Dimethylaminopropyl)-N'-ethylcarbodiimide (EDC) activation
coupling chemistry............................................................................................................ 79
3.3.3 Conjugation via reactive heterobifunctional linkers ......................................................... 80
Content 5
3.3.4 Conjugation of Ki-4 single chain variable fragment......................................................... 84
3.3.5 Conjugation of Ki-4 mouse monoclonal antibody............................................................ 86
3.3.6 Evaluation of binding affinity of antibody siRNA conjugates.......................................... 88
3.3.7 Evaluation of cytotoxic activity........................................................................................ 89
3.3.8 Summary........................................................................................................................... 90
4 Discussion............................................................................................... 91
4.1 siRNA mediated knock down of eukaryotic elongation factor 2.................... 91
4.2 Cell surface receptor mediated siRNA delivery ............................................. 94
4.3 Antibody mediated siRNA delivery ............................................................... 98
5 Outlook .................................................................................................. 102
6 Summary................................................................................................ 104
7 Literature ............................................................................................... 106
8 Appendix 117
8.1 List of abbreviations ..................................................................................... 117
8.2 Publications................................................................................................... 121
8.3 Poster presentations ...................................................................................... 121


1 Introduction
1.1 RNA interference
The phenomenon of gene silencing was initially described in plants. It was discovered
that the introduction of an additional gene