Novel strategies to improve the efficiency of therapeutic adenoviruses for the treatment of cancer [Elektronische Ressource] / vorgelegt von Stanimira Rohmer

Dani Plana Trenchs And Mara Wish

Sujets

• Biologie

Informations

• Publié par: friedrich-alexander-universitat_erlangen-nurnberg
• Publié le: 01 janvier 2010
• Nombre de lectures: 15
• Langue: English
• Poids de l'ouvrage: 2 Mo

Extrait

Novel Strategies to Improve the Efficiency of
Therapeutic Adenoviruses for the
Treatment of Cancer











Den Naturwissenschaftlichen Fakultäten der
Friedrich-Alexander-Universität Erlangen-Nürnberg




zur
Erlangung des Doktorgrades














vorgelegt von
Stanimira Rohmer
aus Nürnberg










Als Dissertation genehmigt von den Naturwissenschaftlichen Fakultäten der
Universität Erlangen-Nürnberg



























Tag der mündlichen Prüfung: 22.01.2010



Vorsitzender der
Promotionskommission: Prof. Dr. Eberhard Bänsch

Erstberichterstatter: Prof. Dr. Thomas Winkler

Zweitberichterstatter: PD Dr. Dirk M. Nettelbeck
















Die Neugier steht immer
an erster Stelle eines Problems,
das gelöst werden will

Galileo Galilei






















Table of Contents
Table of Contents
1 Summary..............................................................................1
2 Introduction .........................................................................3
2.1 Cancer and Cancer Therapy of the Last Centuries............................ 3
2.2 Gene Therapy for Cancer Treatment ................................................... 8
2.3 Virotherapy for Cancer Treatment .....................................................12
2.4 Adenoviruses and their Use as Gene Therapy Vector or Oncolytic
Virus .....................................................................................................14
2.4.1 Adenoviruses: Virion Structure, Cell Entry and Genome Organization....... 15
2.4.1.1 Serotypes and Virus Structure .................................................................................... 15
2.4.1.2 Cell Binding and Entry................................................................................................. 16
2.4.1.3 Genome Organization and Viral Replication ............................................................... 17
2.4.2 Adenoviral Vectors...................................................................................... 20
2.4.2.1 First-Generation Vectors ............................................................................................. 20
2.4.2.2 Second-Generation Vectors........................................................................................ 20
2.4.2.3 Helper-Dependent Vectors.......................................................................................... 21
2.4.2.4 Conditionally Replication-Competent/Oncolytic Adenoviruses ................................... 21
2.4.3 Oncolytic Adenoviruses .............................................................................. 22
2.4.3.1 Tumor-selective Replication and Lysis of Oncolytic Adenoviruses by Viral Gene
Mutations.................................................................................................................... 23
2.4.3.2 ctive Replication and Lysis of Oncolytic Aby Using Tissue-or
Tumor-Selective Promoters 24
2.4.3.3 Genetic Modification of the Virus Capsid for Efficient Cell Entry of Oncolytic
Adenoviruses.............................................................................................................. 25
2.4.3.4 Potential Hurdles Limiting Oncolytic Adenovirus Efficacy........................................... 27
2.4.3.5 Strategies to Improve the Therapeutic Efficacy of Oncolytic Adenoviruses................ 28
3 Objectives of the study ....................................................31
4 Materials and methods .....................................................32
4.1 Materials ...............................................................................................32
4.1.1 Chemicals, filters and enzymes .................................................................. 32
4.1.2 Buffers and solutions .................................................................................. 32
4.1.2.1 Buffers and solutions for gel electrophoresis .............................................................. 32
4.1.2.1.1 Electrophoresis of nucleic acids............................................................................. 32
4.1.2.1.2 phoresis of proteins .................................................................................... 32
4.1.2.2 Buffers and solutions for western blot analysis........................................................... 33
4.1.2.3 Buffers and solutions for flow cytometry ..................................................................... 33
4.1.2.4 for viral lysis.............................................................................. 33
4.1.2.5 Buffers and solutions for production of transformation competent bacteria................ 33
4.1.2.6 s for DNA precipitation ................................................................ 33
4.1.2.7 Buffers and solutions for caesium chloride equilibrium density ultracentrifugation..... 33
4.1.3 Media.......................................................................................................... 34
4.1.3.1 Media for bacterial culture........................................................................................... 34
4.1.3.2 Media and solutions for cell culture............................................................................. 34 Table of Contents
4.1.4 Cells and Bacteria Strains .......................................................................... 34
4.1.4.1 Bacteria strains............................................................................................................ 34
4.1.4.2 Human cells lines ........................................................................................................ 35
4.1.5 Adenoviruses.............................................................................................. 36
4.1.6 Nucleic acids 36
4.1.6.1 Oligonucleotides.......................................................................................................... 36
4.1.6.1.1 Oligonucleotides for PCR cloning 36
4.1.6.1.2 ucleotides for controlling recombinant modified Ad genomes..................... 37
4.1.6.1.3 Oligonuclsequencing............................................................................ 38
4.1.6.1.4 uclquantitative real time PCR (qPCR) ....................................... 38
4.1.6.2 Plasmids ...................................................................................................................... 39
4.1.6.3 Antibodies.................................................................................................................... 40
4.1.6.3.1 s for western blot analysis....................................................................... 40
4.2 Methods................................................................................................41
4.2.1 Nucleic acid methods.................................................................................. 41
4.2.1.1 DNA cloning................................................................................................................. 41
4.2.1.2.1 Production of transformation-competent bacteria and transformation................... 41
4.2.1.2.1.1 Production of chemical-competent bacteria ansformation by heat shock. 41
4.2.1.2.1.2 Production of electro-competent bacteria and tran
electroporation .............................................................................................. 42
4.2.1.2.1.3 Homologous recombination for the generation of recombinant adenoviral
genomes ....................................................................................................... 43
4.2.1.3 Preparation of DNA and RNA...................................................................................... 43
4.2.1.3.1 Analytical isolation of plasmid DNA (mini lysate)................................................... 43
4.2.1.3.2 Quantitative isolation of plasmid DNA (midi lysate)............................................... 43
4.2.1.3.3 DNA isolation from infected human cell cultures 44
4.2.1.3.4 RNA isolation ......................................................................................................... 44
4.2.1.4 PCR (poymerase chain reaction) ................................................................................ 44
4.2.1.4.1 Quantitative real time PCR (qPCR) ....................................................................... 45
4.2.1.5 Protein biochemical and immunological methods....................................................... 46
4.2.1.5.1 Preparation of total cell lysates.............................................................................. 46
4.2.1.5.2 Determination of total protein concentration .....................................................