Modulation of viral gene expression and capsid components to engineer oncolytic adenoviruses for virotherapy of cancer:
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| Beschreibung: | 155 S. Ill., graph. Darst. |
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| 245 | 1 | 0 | |a Modulation of viral gene expression and capsid components to engineer oncolytic adenoviruses for virotherapy of cancer |c von Daniela Kosmides |
| 264 | 1 | |c 2008 | |
| 300 | |a 155 S. |b Ill., graph. Darst. | ||
| 336 | |b txt |2 rdacontent | ||
| 337 | |b n |2 rdamedia | ||
| 338 | |b nc |2 rdacarrier | ||
| 502 | |a Erlangen-Nürnberg, Univ., Diss., 2008 | ||
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Datensatz im Suchindex
| _version_ | 1804137747455148032 |
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| adam_text | Table of Contents
1 Zusammenfassung 1
Summary 2
2 Introduction 3
2.1 Oncolytic adenoviruses: the strategy 3
2.1.1 Viral oncolysis as an emerging approach for molecular treatment of cancer 3
2.1.1.1 Favourable attributes of adenoviruses qualifying them for virotherapy 5
2.1.2 Targeting strategies of CRAds for efficient and selective virotherapy of cancer 5
2.2 Adenovirus capsid structure, cell entry and genome organization 6
2.2.1 Adenovirus capsid structure 6
2.2.2 Adenovirus cell entry 7
2.2.3 Adenovirus genome organization and gene functions 8
2.3 Strategies for restricting Ad replication to tumor cells 10
2.4 Strategies for targeting transduction of adenoviruses 13
2.5 Strategy for enhancing the lytic potency of oncolytic adenoviruses 17
3 Objective of the study 19
4 Materials and methods 20
4.1 Materials 20
4.1.1 Chemicals, filters and enzymes 20
4.1.2 Buffers and solutions 20
4.1.2.1 Buffers and solutions for gel electrophoresis 20
4.1.2.1.1 Electrophoresis of nucleic acids 20
4.1.2.1.2 Electrophoresis of proteins 20
4.1.2.2 Buffers and solutions for western blot analysis 21
4.1.2.3 Buffers and solutions for flow cytometry 21
4.1.2.4 Buffers and solutions for viral lysis 21
4.1.2.5 Buffers and solutions for production of transformation competent bacteria 21
4.1.2.6 Buffers and solutions for DNA precipitation 21
4.1.2.7 Buffers and solutions for caesium chloride equilibrium density ultracentrifugation 22
4.1.3 Media 22
4.1.3.1 Media for bacterial culture 22
4.1.3.2 Media and solutions for cell culture 22
4.1.4 Cells 23
4.1.4.1 Bacteria strains ^
A A.4.2 Human cells lines ^
4.1.5 Adenoviruses
4.1.6 Nucleic acids 26
4.1.6.1 Oligonucleotides
4.1.6.1.1 Oligonucleotides for PCR cloning 2°
4.1.6.1.2 Oligonucleotides for annealing fl
4.1.6.1.3 Oligonucleotides tor controlling recombinant modified Ad genomes 28
4.1.6.1.4 Oligonucleotides for sequencing : 2=*
4.1.6.1.5 Oligonucleotides for semi-quantitative reverse transcriptase polymerase chain
reaction (RT-PCR) 29
4.1.6.1.6 Oligonucleotides for quantitative realtime PCR (qPCR) 30
4.1.6.2 Plasmids 30
4.1.6.3 Antibodies 32
4.1.6.3.1 Antibodies for western blot analysis 3J
4.1.6.3.2 Antibodies for FACS-analysis 3Z
4.1.6.4 Peptides and proteins 33
4.1.7 Methods 33
4.1.7.1 Nucleic acid methods 33
4.1.7.1.1 DNA cloning 33
4.1.7.1.2 Production of transformation-competent bacteria and transformation 34
4.1.7.1.2.1 Production of chemical-competent bacteria and transformation by heat shock.
4.1.7.1.2.2 Production of electro-competent bacteria and transformation by
electroporation 3
4.1.7.1.2.3 Homologous recombination for the generation of recombinant adenoviral
genomes ^
4.1.7.1.3 Preparation of DNA and RNA 35
4.1.7.1.3.1 Analytical isolation of plasmid DNA (mini lysate) ¦
4.1.7.1.3.2 Quantitative isolation of plasmid DNA (midi and maxi lysate) 3°
4.1.7.1.3.3 DNA isolation from infected human cell cultures 3®
4.1.7.1.3.4 RNA isolation and reverse transcription ^°
4.1.7.1.4 Annealing of oligonucleotides ^
4.1.7.1.5 PCR (polymerase chain reaction) 3
4.1.7.1.6 Two step PCR 3°
4.1.7.1.7 Quantitative real time PCR (qPCR) 39
4.1.7.2 Protein biochemical and immunological methods 3^
4.1.7.2.1 Preparation of total cell lysates 3j*
4.1.7.2.2 Determination of total protein concentration :
4.1.7.2.3 Discontinous SDS-Polyacrylamidgelelectrophoresis (SDS-Page) 4°
4.1.7.2.4 Western Transfer . . 40
4.1.7.2.5 Immunoblot 40
4.1.7.2.6 Flow cytometry for the detection of human HER2/Neu surface antigen 41
4.1.7.3 Cell culture 41
4.1.7.3.1 Passaging, freezing and thawing cell culture cells *l
4.1.7.3.2 Growth arrest of primary cells 4
4.1.7.3.3 Transient transfections and reporter assays 0
4.1.7.3.3.1 Transient transfectton for the analysis ot promoter activities
4.1.7.3.3.2 Transient transfection for investigation of trimerization of modified fiber
constructs 43
4.1.7.3.3.3 Luciferase reporter assay 43
4.1.7.3.3.4 P-galactostdase (Pgal) assay .L .[ZZZ . . . . . .. 43
4.1.7.3.4 Crystal violet staining of infected cells 44
4.1.7.4 Recominant adenovirus **¦
4.1.7.4.1 Generation of recombinant adenovitus **
4.1.7.4.2 Generation of viral particles containing recombinant fiber molecules 45
4.1.7.4.3 Caesium chloride gradient equilibrium density ultracentrifugation for the purification
of viral particles 46
4.1.7.4.3.1 Determination of viral particle concentration 46
4.1.7.4.3.1.1 Determination of physical viral particles 46
4.1.7.4.3.1.1.1 Determination of physical viral particles by reading optical density 46
4.1.7.4.3.1.1.2 Determination of physical viral particles by real time PCR 47
4.1.7.4.3.1.2 Determination of infectious particle concentration using the Tissue
Culture Infectious Dose 50 (TCIDsoj-assay 47
4.1.7.4.3.2 Verification of recombinant adenoviral genomes 48
4.1.7.4.3.3 Transduction and infections of cells with recombinant adenovirus 48
4.1.7.4.3.3.1 Transduction with replication-deficient adenovirus for the analysis of
luciferase activities 48
4.1.7.4.3.3.2 Infection with replication-competent adenovirus for the analysis of E1A-
expression 48
4.1.7.4.3.3.3 Infection for cytotoxicity assay 49
4.1.7.4.3.3.4 Infection for quantification of adenoviral genomes by qPCR 49
4.1.7.4.3.3.5 Transduction for gene transfer assay with fiber modified replication-
deficient Ad 49
4.1.7.4.3.3.6 Transduction for receptor blocking assay 50
5 Results 51
5.1 Comparative evaluation of tumor-specific pan-cancer promoters for transcriptional
targeting of adenoviral oncolysis to cancer 51
5.1.1 Investigation of tumor marker mRNA expression profiles in melanoma and primary
normal cells by semi-quantitative RT-PCR 52
5.1.2 Analysis of Survivin, E2F-1 and TERT promoter activity and specificity in various
cancer cells and primary normal control cells 55
5.1.2.1 Construction of recombinant luciferase reporter gene plasmids 55
5.1.2.2 Analysis of Surv and TERT promoter activities in human melanoma and primary
normal control cells by transient transfection of luciferase reporter gene plasmids.... 56
5.1.2.3 Construction of replication-deficient Ad incorporating TSP 59
5.1.2.4 Activities of Surv, E2F-1 and TERT promoters in rdAd driving luciferase reporter gene
expression in various tumor cell lines and primary normal control cells 61
5.1.3 Construction of replication-competent Ad incorporating TSP to drive expression of E1A... 65
5.1.4 Analysis of ElA-expression after infection of various tumor and primary normal cells with
recombinant CRAds 69
5.1.5 Analysis of genome replication of oncolytic adenoviruses in tumor cells and primary normal
control cells 72
5.1.6 Investigation of the cytotoxicity of oncolytic adenoviruses in a panel of tumor cells and
primary normal control cells 75
5.2 Tropism modification of Adenoviruses by ligand insertion into various positions of the
Ad serotype 41 short-fiber knob domain 78
5.2.1 Generation of expression plasmids for chimeric fibers with inserted RGD peptides 79
5.2.2 Analysis of trimerization of recombinant fiber molecules containing the Ad41s fiber knob
with RGD peptide insertions 81
5.2.3 Investigation of incorporation of recombinant fiber molecules containing the Ad41s fiber
knob with RGD peptide into virus particles 83
5.2.4 Transduction efficiencies of adenovirus containing Ad41 s fiber-derived molecules with RGD
peptide insertions in the knob domain 84
5.2.5 Competition of transduction by fiber-modified adenoviruses with soluble RGD peptide 86
5.2.6 Fiber trimerization and adenovirus transduction by F5/41 sU-RGD derivatives with a long
fiber shaft, with tandem copies of the peptide insert, or with a longer peptide insert 88
5.2.7 Tropism modified Adenoviruses incorporating an Affibody molecule specific for the tumor
antigen HER2/Neu into various positions of the Ad serotype 41 short-fiber knob domain .. 91
5.2.8 Generation of recombinant fiber expression plasmids with inserted HER2/Neu Affibody
molecules 91
5.2.9 Analysis of trimerization of recombinant fiber molecules containing the Ad41s fiber knob
with HER2/Neu specific Affibody molecule and incorporation into virus particles 93
5.2.10 Evaluation of HER2/Neu surface protein expression in a panel of cancer cells 94
5.2.11 Transduction efficiency of adenovirus containing Ad41s fiber-derived molecules with
HER2/Neu Affibody fused to C-terminus of the knob domain 96
6 Discussion 99
6.1 Transcriptional targeting oi CRAds
6.1.1 Investigation oi tumor marker mRNA expression profiles 1^1
6.1.2 Analysis of pan-cancer promoter activities and specificities in various cancer cells and
primary normal control cells - 1^2
6.1.2.1 Pan-cancer promoter analysis by transient transfection of luciferase reporter gene
plasmids 102
6.1.2.2 Pan-cancer promoter activities in rdAd driving luciferase reporter gene 104
6.1.3 Analysis of CRAds targeted by TSP for adenoviral oncolysis 106
6.1.3.1 Analysis of E1 A-expression after infection with recombinant TSP-CRAds 1°7
6.1.3.2 Analysis of genome replication following infection with recombinant TSP-CRAds... 110
6.1.3.3 Analysis of cytopathic effect mediated by recombinant TSP-CRAds 1^2
6.1.3.3.1 Cytotoxicity in tumor cells 112
6.1.3.3.2 Selectivity of cell killing by TSP-CRAds 115
6.1.3.4 Conclusions for the development of pan-cancer targeted TSP-CRAds 116
6.2 Transductional targeting of CRAds 119
6.2.1 Trimerization and incorporation of recombinant fibers containing the Ad41 s fiber knob with
the RGD peptide into adenoviruses 12
6.2.2 Transduction efficacies of adenoviruses with recombinant fibers containing RGD peptides
insertion in the knob domain 122
6.2.3 Competition of transduction by fiber-modified adenoviruses with soluble RGD peptide.... 124
6.2.4 Tropism modified Adenoviruses incorporating an Affibody molecule specific for the tumor
antigen HER2/Neu into various positions of the Ad serotype 41 short-fiber knob domain 126
6.2.4.1 Trimerization of recombinant F5/F41S fibers containing HER2/Neu Affibody molecule
and incorporation into adenoviruses ^
6.2.4.2 Transduction experiments of Ad incorporating HER2/Neu Affibody molecule I28
7 References 132
8 Table of Figures 147
9 Abbrevations 149
10 Attachments 152
11 Acknowledgements 153
12 Curriculum Vitae 154
13 Publications 155
8 Table of Figures
Fig. 1: Oncolytic viral spread 4
Fig. 2: Schematic representation of the Ad5 virion structure 7
Fig. 3: Schematic representation of adenovirus genome and transcription units: 10
Fig. 4: Transcriptional targeting of viral replication using tissue or tumor-specific
promoters 11
Fig. 5: Targeting Ad5 cell attachment 14
Fig. 6: Investigation of tumor marker mRNA expression profiles by semi-quantitative
RT-PCR 53
Fig. 7: Evaluation of tumor marker mRNA expression in growth arrested normal
control cells 55
Fig. 8: Schematic representation of different Survivin constructs 56
Fig. 9: Analysis of promoter activities in human melanoma and primary normal
control cells by transient transfection 57
Fig. 10: Schematic representation of homologous recombination for the construction
of replication-deficient Ad genomes 60
Fig. 11: Luciferase activities in various tumor cell lines and primary normal control
cells after transduction with Ad vectors bearing promoter-constructs driving
reporter gene 63
Fig. 12: Analysis of Surv and E2F-1 promoter activities after adenoviral transduction
of growth arrested primary normal control cells 65
Fig. 13: Schematic representation of genome composition of recombinant oncolytic
Ad generated by introducing different modifications into the Ad5 genome.... 66
Fig. 14: Schematic representation of modified plasmids employed for the construction
of recombinant CRAds: 68
Fig. 15: Western blot analysis of E1A expression after infection of various tumor cell
lines and primary normal control cells with promoter controlled CRAds 71
Fig. 16: Analysis of genome replication of oncolytic adenoviruses in tumor cell lines
and primary normal control cells 74
Fig. 17: Investigation of cytopathic effect of replication-competent adenoviruses in a
panel of tumor cell lines and primary normal control cells by crystal violet
staining 77
Fig. 18: Outline of RGD peptide insertions into various positions of the Ad41s fiber
knob 80
Fig. 19: Trimerization of recombinant Ad41s fibers and incorporation into virus
particles 82
Fig. 20: Transduction of established and primary melanoma cells and of normal cells
with fiber-modified adenoviruses 85
Fig. 21: Competition of transduction by fiber-modified adenoviruses with soluble RGD
peptide 87
Fig. 22: Outline of pF5/41slJ-RGD derivatives with a long fiber shaft, with two copies
of RGD peptide insert in tandem, or with an extended RGD-containing peptide
insert 88
Fig. 23: Analysis of pF5/41slJ-RGD derivatives with a long shaft, with two copies of
the RGD peptide insert in tandem, or with extended RGD-containing peptide
insert 90
Fig. 24: Outline of HER2/Neu Affibody molecule insertions into various positions of
the Ad41s fiber knob 92
Fig. 25: Trimerization of recombinant F5/41s fibers carrying a HER2/Neu specific
Affibody molecule and incorporation into virus particles 94
Fig. 26: Evaluation of HER2/Neu surface protein expression on tumor cell lines 95
Fig. 27: Transduction of various tumor cell lines with fiber-modified adenoviruses
carrying HER2/Neu specific Affibody molecule 97
Fig. 28: Competition of transduction by fiber-modified adenoviruses carrying
HER2/Neu-specific Affibody with soluble HER2/Neu or HER2/Neu-binding
Affibody molecule 98
|
| adam_txt |
Table of Contents
1 Zusammenfassung 1
Summary 2
2 Introduction 3
2.1 Oncolytic adenoviruses: the strategy 3
2.1.1 Viral oncolysis as an emerging approach for molecular treatment of cancer 3
2.1.1.1 Favourable attributes of adenoviruses qualifying them for virotherapy 5
2.1.2 Targeting strategies of CRAds for efficient and selective virotherapy of cancer 5
2.2 Adenovirus capsid structure, cell entry and genome organization 6
2.2.1 Adenovirus capsid structure 6
2.2.2 Adenovirus cell entry 7
2.2.3 Adenovirus genome organization and gene functions 8
2.3 Strategies for restricting Ad replication to tumor cells 10
2.4 Strategies for targeting transduction of adenoviruses 13
2.5 Strategy for enhancing the lytic potency of oncolytic adenoviruses 17
3 Objective of the study 19
4 Materials and methods 20
4.1 Materials 20
4.1.1 Chemicals, filters and enzymes 20
4.1.2 Buffers and solutions 20
4.1.2.1 Buffers and solutions for gel electrophoresis 20
4.1.2.1.1 Electrophoresis of nucleic acids 20
4.1.2.1.2 Electrophoresis of proteins 20
4.1.2.2 Buffers and solutions for western blot analysis 21
4.1.2.3 Buffers and solutions for flow cytometry 21
4.1.2.4 Buffers and solutions for viral lysis 21
4.1.2.5 Buffers and solutions for production of transformation competent bacteria 21
4.1.2.6 Buffers and solutions for DNA precipitation 21
4.1.2.7 Buffers and solutions for caesium chloride equilibrium density ultracentrifugation 22
4.1.3 Media 22
4.1.3.1 Media for bacterial culture 22
4.1.3.2 Media and solutions for cell culture 22
4.1.4 Cells 23
4.1.4.1 Bacteria strains ^
A A.4.2 Human cells lines ^
4.1.5 Adenoviruses
4.1.6 Nucleic acids 26
4.1.6.1 Oligonucleotides "
4.1.6.1.1 Oligonucleotides for PCR cloning 2°
4.1.6.1.2 Oligonucleotides for annealing fl
4.1.6.1.3 Oligonucleotides tor controlling recombinant modified Ad genomes 28
4.1.6.1.4 Oligonucleotides for sequencing : 2=*
4.1.6.1.5 Oligonucleotides for semi-quantitative reverse transcriptase polymerase chain
reaction (RT-PCR) 29
4.1.6.1.6 Oligonucleotides for quantitative realtime PCR (qPCR) 30
4.1.6.2 Plasmids 30
4.1.6.3 Antibodies 32
4.1.6.3.1 Antibodies for western blot analysis 3J
4.1.6.3.2 Antibodies for FACS-analysis 3Z
4.1.6.4 Peptides and proteins 33
4.1.7 Methods 33
4.1.7.1 Nucleic acid methods 33
4.1.7.1.1 DNA cloning 33
4.1.7.1.2 Production of transformation-competent bacteria and transformation 34
4.1.7.1.2.1 Production of chemical-competent bacteria and transformation by heat shock.
4.1.7.1.2.2 Production of electro-competent bacteria and transformation by
electroporation 3
4.1.7.1.2.3 Homologous recombination for the generation of recombinant adenoviral
genomes ^
4.1.7.1.3 Preparation of DNA and RNA 35
4.1.7.1.3.1 Analytical isolation of plasmid DNA (mini lysate) ¦"
4.1.7.1.3.2 Quantitative isolation of plasmid DNA (midi and maxi lysate) 3°
4.1.7.1.3.3 DNA isolation from infected human cell cultures 3®
4.1.7.1.3.4 RNA isolation and reverse transcription ^°
4.1.7.1.4 Annealing of oligonucleotides ^
4.1.7.1.5 PCR (polymerase chain reaction) 3
4.1.7.1.6 Two step PCR 3°
4.1.7.1.7 Quantitative real time PCR (qPCR) 39
4.1.7.2 Protein biochemical and immunological methods 3^
4.1.7.2.1 Preparation of total cell lysates 3j*
4.1.7.2.2 Determination of total protein concentration ":
4.1.7.2.3 Discontinous SDS-Polyacrylamidgelelectrophoresis (SDS-Page) 4°
4.1.7.2.4 Western Transfer . . 40
4.1.7.2.5 Immunoblot 40
4.1.7.2.6 Flow cytometry for the detection of human HER2/Neu surface antigen 41
4.1.7.3 Cell culture 41
4.1.7.3.1 Passaging, freezing and thawing cell culture cells *l
4.1.7.3.2 Growth arrest of primary cells 4
4.1.7.3.3 Transient transfections and reporter assays 0
4.1.7.3.3.1 Transient transfectton for the analysis ot promoter activities
4.1.7.3.3.2 Transient transfection for investigation of trimerization of modified fiber
constructs 43
4.1.7.3.3.3 Luciferase reporter assay 43
4.1.7.3.3.4 P-galactostdase (Pgal) assay \.L'.[ZZZ'.".".'.'.'. 43
4.1.7.3.4 Crystal violet staining of infected cells 44
4.1.7.4 Recominant adenovirus **¦
4.1.7.4.1 Generation of recombinant adenovitus **
4.1.7.4.2 Generation of viral particles containing recombinant fiber molecules 45
4.1.7.4.3 Caesium chloride gradient equilibrium density ultracentrifugation for the purification
of viral particles 46
4.1.7.4.3.1 Determination of viral particle concentration 46
4.1.7.4.3.1.1 Determination of physical viral particles 46
4.1.7.4.3.1.1.1 Determination of physical viral particles by reading optical density 46
4.1.7.4.3.1.1.2 Determination of physical viral particles by real time PCR 47
4.1.7.4.3.1.2 Determination of infectious particle concentration using the Tissue
Culture Infectious Dose 50 (TCIDsoj-assay 47
4.1.7.4.3.2 Verification of recombinant adenoviral genomes 48
4.1.7.4.3.3 Transduction and infections of cells with recombinant adenovirus 48
4.1.7.4.3.3.1 Transduction with replication-deficient adenovirus for the analysis of
luciferase activities 48
4.1.7.4.3.3.2 Infection with replication-competent adenovirus for the analysis of E1A-
expression 48
4.1.7.4.3.3.3 Infection for cytotoxicity assay 49
4.1.7.4.3.3.4 Infection for quantification of adenoviral genomes by qPCR 49
4.1.7.4.3.3.5 Transduction for gene transfer assay with fiber modified replication-
deficient Ad 49
4.1.7.4.3.3.6 Transduction for receptor blocking assay 50
5 Results 51
5.1 Comparative evaluation of tumor-specific "pan-cancer" promoters for transcriptional
targeting of adenoviral oncolysis to cancer 51
5.1.1 Investigation of tumor marker mRNA expression profiles in melanoma and primary
normal cells by semi-quantitative RT-PCR 52
5.1.2 Analysis of Survivin, E2F-1 and TERT promoter activity and specificity in various
cancer cells and primary normal control cells 55
5.1.2.1 Construction of recombinant luciferase reporter gene plasmids 55
5.1.2.2 Analysis of Surv and TERT promoter activities in human melanoma and primary
normal control cells by transient transfection of luciferase reporter gene plasmids. 56
5.1.2.3 Construction of replication-deficient Ad incorporating TSP 59
5.1.2.4 Activities of Surv, E2F-1 and TERT promoters in rdAd driving luciferase reporter gene
expression in various tumor cell lines and primary normal control cells 61
5.1.3 Construction of replication-competent Ad incorporating TSP to drive expression of E1A. 65
5.1.4 Analysis of ElA-expression after infection of various tumor and primary normal cells with
recombinant CRAds 69
5.1.5 Analysis of genome replication of oncolytic adenoviruses in tumor cells and primary normal
control cells 72
5.1.6 Investigation of the cytotoxicity of oncolytic adenoviruses in a panel of tumor cells and
primary normal control cells 75
5.2 Tropism modification of Adenoviruses by ligand insertion into various positions of the
Ad serotype 41 short-fiber knob domain 78
5.2.1 Generation of expression plasmids for chimeric fibers with inserted RGD peptides 79
5.2.2 Analysis of trimerization of recombinant fiber molecules containing the Ad41s fiber knob
with RGD peptide insertions 81
5.2.3 Investigation of incorporation of recombinant fiber molecules containing the Ad41s fiber
knob with RGD peptide into virus particles 83
5.2.4 Transduction efficiencies of adenovirus containing Ad41 s fiber-derived molecules with RGD
peptide insertions in the knob domain 84
5.2.5 Competition of transduction by fiber-modified adenoviruses with soluble RGD peptide 86
5.2.6 Fiber trimerization and adenovirus transduction by F5/41 sU-RGD derivatives with a long
fiber shaft, with tandem copies of the peptide insert, or with a longer peptide insert 88
5.2.7 Tropism modified Adenoviruses incorporating an Affibody molecule specific for the tumor
antigen HER2/Neu into various positions of the Ad serotype 41 short-fiber knob domain . 91
5.2.8 Generation of recombinant fiber expression plasmids with inserted HER2/Neu Affibody
molecules 91
5.2.9 Analysis of trimerization of recombinant fiber molecules containing the Ad41s fiber knob
with HER2/Neu specific Affibody molecule and incorporation into virus particles 93
5.2.10 Evaluation of HER2/Neu surface protein expression in a panel of cancer cells 94
5.2.11 Transduction efficiency of adenovirus containing Ad41s fiber-derived molecules with
HER2/Neu Affibody fused to C-terminus of the knob domain 96
6 Discussion 99
6.1 Transcriptional targeting oi CRAds "
6.1.1 Investigation oi tumor marker mRNA expression profiles 1^1
6.1.2 Analysis of "pan-cancer" promoter activities and specificities in various cancer cells and
primary normal control cells - 1^2
6.1.2.1 "Pan-cancer" promoter analysis by transient transfection of luciferase reporter gene
plasmids 102
6.1.2.2 "Pan-cancer" promoter activities in rdAd driving luciferase reporter gene 104
6.1.3 Analysis of CRAds targeted by TSP for adenoviral oncolysis 106
6.1.3.1 Analysis of E1 A-expression after infection with recombinant TSP-CRAds 1°7
6.1.3.2 Analysis of genome replication following infection with recombinant TSP-CRAds. 110
6.1.3.3 Analysis of cytopathic effect mediated by recombinant TSP-CRAds 1^2
6.1.3.3.1 Cytotoxicity in tumor cells 112
6.1.3.3.2 Selectivity of cell killing by TSP-CRAds 115
6.1.3.4 Conclusions for the development of "pan-cancer" targeted TSP-CRAds 116
6.2 Transductional targeting of CRAds 119
6.2.1 Trimerization and incorporation of recombinant fibers containing the Ad41 s fiber knob with
the RGD peptide into adenoviruses 12"
6.2.2 Transduction efficacies of adenoviruses with recombinant fibers containing RGD peptides
insertion in the knob domain 122
6.2.3 Competition of transduction by fiber-modified adenoviruses with soluble RGD peptide. 124
6.2.4 Tropism modified Adenoviruses incorporating an Affibody molecule specific for the tumor
antigen HER2/Neu into various positions of the Ad serotype 41 short-fiber knob domain 126
6.2.4.1 Trimerization of recombinant F5/F41S fibers containing HER2/Neu Affibody molecule
and incorporation into adenoviruses ^
6.2.4.2 Transduction experiments of Ad incorporating HER2/Neu Affibody molecule I28
7 References 132
8 Table of Figures 147
9 Abbrevations 149
10 Attachments 152
11 Acknowledgements 153
12 Curriculum Vitae 154
13 Publications 155
8 Table of Figures
Fig. 1: Oncolytic viral spread 4
Fig. 2: Schematic representation of the Ad5 virion structure 7
Fig. 3: Schematic representation of adenovirus genome and transcription units: 10
Fig. 4: Transcriptional targeting of viral replication using tissue or tumor-specific
promoters 11
Fig. 5: Targeting Ad5 cell attachment 14
Fig. 6: Investigation of tumor marker mRNA expression profiles by semi-quantitative
RT-PCR 53
Fig. 7: Evaluation of tumor marker mRNA expression in growth arrested normal
control cells 55
Fig. 8: Schematic representation of different Survivin constructs 56
Fig. 9: Analysis of promoter activities in human melanoma and primary normal
control cells by transient transfection 57
Fig. 10: Schematic representation of homologous recombination for the construction
of replication-deficient Ad genomes 60
Fig. 11: Luciferase activities in various tumor cell lines and primary normal control
cells after transduction with Ad vectors bearing promoter-constructs driving
reporter gene 63
Fig. 12: Analysis of Surv and E2F-1 promoter activities after adenoviral transduction
of growth arrested primary normal control cells 65
Fig. 13: Schematic representation of genome composition of recombinant oncolytic
Ad generated by introducing different modifications into the Ad5 genome. 66
Fig. 14: Schematic representation of modified plasmids employed for the construction
of recombinant CRAds: 68
Fig. 15: Western blot analysis of E1A expression after infection of various tumor cell
lines and primary normal control cells with promoter controlled CRAds 71
Fig. 16: Analysis of genome replication of oncolytic adenoviruses in tumor cell lines
and primary normal control cells 74
Fig. 17: Investigation of cytopathic effect of replication-competent adenoviruses in a
panel of tumor cell lines and primary normal control cells by crystal violet
staining 77
Fig. 18: Outline of RGD peptide insertions into various positions of the Ad41s fiber
knob 80
Fig. 19: Trimerization of recombinant Ad41s fibers and incorporation into virus
particles 82
Fig. 20: Transduction of established and primary melanoma cells and of normal cells
with fiber-modified adenoviruses 85
Fig. 21: Competition of transduction by fiber-modified adenoviruses with soluble RGD
peptide 87
Fig. 22: Outline of pF5/41slJ-RGD derivatives with a long fiber shaft, with two copies
of RGD peptide insert in tandem, or with an extended RGD-containing peptide
insert 88
Fig. 23: Analysis of pF5/41slJ-RGD derivatives with a long shaft, with two copies of
the RGD peptide insert in tandem, or with extended RGD-containing peptide
insert 90
Fig. 24: Outline of HER2/Neu Affibody molecule insertions into various positions of
the Ad41s fiber knob 92
Fig. 25: Trimerization of recombinant F5/41s fibers carrying a HER2/Neu specific
Affibody molecule and incorporation into virus particles 94
Fig. 26: Evaluation of HER2/Neu surface protein expression on tumor cell lines 95
Fig. 27: Transduction of various tumor cell lines with fiber-modified adenoviruses
carrying HER2/Neu specific Affibody molecule 97
Fig. 28: Competition of transduction by fiber-modified adenoviruses carrying
HER2/Neu-specific Affibody with soluble HER2/Neu or HER2/Neu-binding
Affibody molecule 98 |
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| author | Kosmides, Daniela 1976- |
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| author_role | aut |
| author_sort | Kosmides, Daniela 1976- |
| author_variant | d k dk |
| building | Verbundindex |
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| ctrlnum | (OCoLC)246686642 (DE-599)BVBBV023376925 |
| dewey-full | 615.3 |
| dewey-hundreds | 600 - Technology (Applied sciences) |
| dewey-ones | 615 - Pharmacology and therapeutics |
| dewey-raw | 615.3 |
| dewey-search | 615.3 |
| dewey-sort | 3615.3 |
| dewey-tens | 610 - Medicine and health |
| discipline | Medizin |
| discipline_str_mv | Medizin |
| format | Thesis Book |
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| spelling | Kosmides, Daniela 1976- Verfasser (DE-588)135842719 aut Modulation of viral gene expression and capsid components to engineer oncolytic adenoviruses for virotherapy of cancer von Daniela Kosmides 2008 155 S. Ill., graph. Darst. txt rdacontent n rdamedia nc rdacarrier Erlangen-Nürnberg, Univ., Diss., 2008 Langzeitarchivierung Nationalbibliothek gewährleistet Adenoviren (DE-588)4141412-3 gnd rswk-swf Onkolyse (DE-588)4172588-8 gnd rswk-swf (DE-588)4113937-9 Hochschulschrift gnd-content Adenoviren (DE-588)4141412-3 s Onkolyse (DE-588)4172588-8 s DE-604 Erscheint auch als Online-Ausgabe urn:nbn:de:bvb:29-opus-9721 https://open.fau.de/handle/openfau/654 Verlag kostenfrei Volltext https://nbn-resolving.org/urn:nbn:de:bvb:29-opus-9721 Resolvingsystem http://d-nb.info/989553698/34 HBZ Datenaustausch application/pdf http://bvbr.bib-bvb.de:8991/F?func=service&doc_library=BVB01&local_base=BVB01&doc_number=016560091&sequence=000002&line_number=0001&func_code=DB_RECORDS&service_type=MEDIA Inhaltsverzeichnis |
| spellingShingle | Kosmides, Daniela 1976- Modulation of viral gene expression and capsid components to engineer oncolytic adenoviruses for virotherapy of cancer Adenoviren (DE-588)4141412-3 gnd Onkolyse (DE-588)4172588-8 gnd |
| subject_GND | (DE-588)4141412-3 (DE-588)4172588-8 (DE-588)4113937-9 |
| title | Modulation of viral gene expression and capsid components to engineer oncolytic adenoviruses for virotherapy of cancer |
| title_auth | Modulation of viral gene expression and capsid components to engineer oncolytic adenoviruses for virotherapy of cancer |
| title_exact_search | Modulation of viral gene expression and capsid components to engineer oncolytic adenoviruses for virotherapy of cancer |
| title_exact_search_txtP | Modulation of viral gene expression and capsid components to engineer oncolytic adenoviruses for virotherapy of cancer |
| title_full | Modulation of viral gene expression and capsid components to engineer oncolytic adenoviruses for virotherapy of cancer von Daniela Kosmides |
| title_fullStr | Modulation of viral gene expression and capsid components to engineer oncolytic adenoviruses for virotherapy of cancer von Daniela Kosmides |
| title_full_unstemmed | Modulation of viral gene expression and capsid components to engineer oncolytic adenoviruses for virotherapy of cancer von Daniela Kosmides |
| title_short | Modulation of viral gene expression and capsid components to engineer oncolytic adenoviruses for virotherapy of cancer |
| title_sort | modulation of viral gene expression and capsid components to engineer oncolytic adenoviruses for virotherapy of cancer |
| topic | Adenoviren (DE-588)4141412-3 gnd Onkolyse (DE-588)4172588-8 gnd |
| topic_facet | Adenoviren Onkolyse Hochschulschrift |
| url | https://open.fau.de/handle/openfau/654 https://nbn-resolving.org/urn:nbn:de:bvb:29-opus-9721 http://d-nb.info/989553698/34 http://bvbr.bib-bvb.de:8991/F?func=service&doc_library=BVB01&local_base=BVB01&doc_number=016560091&sequence=000002&line_number=0001&func_code=DB_RECORDS&service_type=MEDIA |
| work_keys_str_mv | AT kosmidesdaniela modulationofviralgeneexpressionandcapsidcomponentstoengineeroncolyticadenovirusesforvirotherapyofcancer |