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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 .......................................................... 46
4.2.1.5.3 Discontinous SDS-Polyacrylamidgelelectrophoresis (SDS-Page) ........................ 46
4.2.1.5.4 Western Transfer ................................................................................................... 46
4.2.1.5.5 Immunoblot ............................................................................................................ 47
4.2.1.5.6 Flow cytometry for the detection of cell death 47
4.2.1.6 Cell culture................................................................................................................... 48
4.2.1.6.1 Passaging, freezing and thawing cell culture cells ................................................ 48
4.2.1.6.2 Transient transfections and reporter assays.......................................................... 48
4.2.1.6.2.1 Transient transfection for the analysis of promoter activities ......................... 48
4.2.1.6.2.2 Luciferase reporter assay ............................................................................... 49
4.2.1.7 Recominant adenovirus............................................................................................... 49
4.2.1.7.1 Generation of recombinant adenovirus.................................................................. 49
4.2.1.7.2 Caesium chloride gradient equilibrium density ultracentrifugation for the purification
of viral particles.................................................................................................... 50
4.2.1.7.3 Determination of viral particle concentration.......................................................... 51
4.2.1.7.3.1 n of infectious particle concentration using the Tissue Culture
Infectious Dose 50 (TCID )-assay............................................................... 51 50
4.2.1.7.3.2 Determination of physical viral particles by reading optical density ............... 52
4.2.1.7.3.3 Verification of recombinant adenoviral genomes ........................................... 52
4.2.1.7.4 Transduction and infections of cells with recombinant adenovirus........................ 52
4.2.1.7.4.1 Transduction with replication-deficient adenovirus for the analysis of luciferase
activities ........................................................................................................ 52 Table of Contents
4.2.1.7.4.2 Ad5wt superinfection after transfection .......................................................... 52
4.2.1.7.4.3 Co-infection assay .......................................................................................... 53
4.2.1.7.4.4 Infection with replication-competent adenovirus with subsequent inhibition of
virus genome replication by AraC................................................................. 53
4.2.1.7.4.5 Infection for cytotoxicity assay........................................................................ 53
4.2.1.7.4.6 Crystal violet staining of infected cells............................................................ 53
4.2.1.7.4.7 Infection for quantification of adenoviral mRNA or adenoviral genomes by
qPCR ............................................................................................................ 53
4.2.1.7.4.8 Infection for the analysis of protein-expression .............................................. 54
4.2.1.7.4.9 Infection for the quantification of infectious particles of oncolytic adenoviruses
w / w/o adding Z-VAD-FMK or Q-VD-OPH................................................... 54
5 Results ...............................................................................55
5.1 Heat-regulated transgene expression from either replication-
deficient or replication-competent adenoviral vectors ...................55
5.1.1 Analysis of heat-inducibility of promoter fragments of stress-inducible genes
................................................................................................................... 55
5.1.2 Influence of adenovirus co-infection and replication on hsp70B’ promoter
activity in a reporter plasmid ....................................................................... 57
5.1.3 Regulation of transgene expression by the hsp70B’ promoter in a
replication-deficient adenovirus vector........................................................ 58
5.1.4 Activity and regulation of the hsp70B’ promoter in an oncolytic adenovirus 60
5.1.5 Insulator elements as tools for improved regulation of the hsp70B’ promoter
in replication-deficient but not replicating adenoviral vectors...................... 67
5.2 Combining Rational Mutagenesis and Transgene Expression for
Improving Efficacy of Oncolytic Adenoviruses ...............................70
5.2.1 Spread of E1B19K-deleted, transgene-encoding oncolytic adenovirus in
tumor cell cultures....................................................................................... 70
5.2.2 Apoptosis induction and cell type-dependent modulation of the adenovirus
replication cycle by the E1B19K-deletion.................................................... 72
5.2.3 Modulation of the expression of transgenes inserted into oncolytic
adenoviruses by E1B19K deletion.............................................................. 79
6 Discussion.........................................................................82
6.1 Insulated hsp70B’ promoter: stringent heat-inducible activity in
replication-deficient, but not replication-competent adenoviruses82
6.1.1 Feasibility of the hsp70B’ promoter fragment for heat-inducible transgene
expression and its analysis in the context of a replication-deficient
adenoviral vector ........................................................................................ 82
6.1.2 Analysis of heat-directed transgene expression in the context of a
replication-competent/oncolytic adenoviral vector ...................................... 87
6.2 Transgene Expression by Oncolytic Adenoviruses is Modulated by
E1B19K-Deletion in a Cell Type-Dependent Manner .......................91
7 References.........................................................................95 Table of Contents
8 Abbreviations ..................................................................109
9 Curriculum Vitae .............................................................112
10 Acknowledgements/Danksagung..................................113
11 Publications.....................................................................114 Zusammenfassung 1
1 ZUSAMMENFASSUNG
Nicht zuletzt die Resistenz vieler Tumoren gegenüber konventionellen Therapien,
begründet die Suche nach neuen Strategien zur Krebsbekämpfung. Zwei innovative
Krebstherapien sind Gentherapie und Virotherapie. Ziel der Gentherapie ist es,
therapeutische Gene, vorwiegend mittels viraler Vektoren, in Tumorzellen
einzufügen, um diese selektiv zu töten. In der Virotherapie dagegen, werden
Tumorzellen mit tumorspezifisch replikationsfähigen Viren (=onkolytische Viren)
infiziert und durch deren lytischen Replikationszyklus zerstört.
Erfolgreiche Gentherapieansätze erfordern effiziente und zielgerichtete Gentransfer-
Vektoren. Therapeutischer Gentransfer mittels replikationsdefizienter und neuerdings
auch mit Hilfe replikationskompetenter/onkolytischer Adenoviren (OAds) erwies sich
als vielversprechend. Spezielle Anwendungen können jedoch eine externe Kontrolle
der Transgenexpression erfordern. Hierzu analysierte das erste Projekt erstmals ein
245bp Fragment des humanen hsp70B’ Promoters bezüglich seiner Fähigkeit Hitze-
regulierbare Transgenexpression im Kontext von replikations-defizienten oder
onkolytischen Ads zu vermitteln. In Transfektionsstudien zeigte dieser Promoter
geringe basale Aktivität; nach Hitze-Induktion stieg die Aktivität dagegen bis zu 3741-
fach an. Nach Insertion des hsp70B’ Promoters in ein nicht-replizierendes Ad, führte
Hitzeschock zu einer 586-fachen höheren Luziferase Aktivität in SK-MEL-28 Zellen;
diese war in HeLa Zellen, aufgrund höherer basaler Aktivität, nur 44-fach. Zur
Verbesserung der Hitze-Induzierbarkeit wurden zwei neue Ad Vektoren generiert, die
mit Hilfe von Isolatoren oberhalb des hsp70B’ Promoters zu einer stringenteren
Hitze-induzierbaren Genexpression mit bis zu 8000-fachen Induktionsraten führte.
Dagegen in einem melanom-spezifischen OAd war die Regulation des hsp70B’
Promoters, insbesondere während der späten Virus-Replikation, aufgehoben und
konnte auch durch Isolatoren nicht wiederhergestellt werden. Die Studie verdeutlicht
somit, dass sich Ad Vektoren mit isoliertem hsp70B’ Promoter speziell in
Gentherapien einsetzen ließen, die eine externe Kontrolle von Transgenen erfordern.
Zudem zeigt sie, dass die Replikation von Vektoren zur Deregulation inserierter
Promotoren führen kann, was bei zukünftigen Entwicklungen transkriptionell
regulierter onkolytischer Gentransfer-Vektoren berücksichtigt werden sollte.
Adenoviren wurden auch vielfach in Virotherapien eingesetzt. Klinische Studien
haben das Potential und die Sicherheit von OAds bewiesen, aber auch deren
unzureichende therapeutische Wirksamkeit. Ziel des zweiten Projekts war es, durch
Kombination zweier Strategien die therapeutische Effizienz von OAds zu steigern:
Die Deletion des anti-apoptotischen, frühen viralen Gens E1B19K, die auf
beschleunigte Virusstreuung durch frühere Virenfreisetzung aus Tumorzellen
abzielte, wurde mit der Insertion von Transgenen in die späte virale
Transkriptionseinheit kombiniert. Bis dato war nicht bekannt, ob sich vorzeitige
Tumorzelllyse nachteilig auf die späte Transgenexpression auswirkt. Analysen der
E1B19K-Deletion resultierten in einigen Tumorzellkulturen in verstärkter früher
Virusfreisetzung und gesteigerter Zelllyse, in anderen dagegen in reduzierter
Onkolyse. Diese Diskrepanz war jedoch nicht auf mutationsbedingte Unterschiede in
der Apoptose-Induktion zurückzuführen. Vielmehr bedingte die E1B19K-Deletion
eine beschleunigte Virusfreisetzung bzw. reduzierte Virusreplikation und Onkolyse.
Erstere hatte jedoch keine nachteilige Wirkung auf die späte Transgenexpression,
die dadurch eher verbessert wurde und für das E1B19K- Virus eine um 3 log-Stufen
gesteigerte Onkolyse und Transgenexpression in A549 Zellen ergab. Somit zeigt die
Studie, dass frühe Virusfreisetzung nicht nur die Virusstreuung und damit die
Onkolyse beschleunigt, sondern dass sie gleichzeitig die Transgenexpression
verstärkt. Im Hinblick auf die rasche Neutralisierung durch anti-virale Immunität,
könnte ein solch beschleunigter Phänotyp die Effizienz von transgentragenden OAds
wesentlich verbessern. Summary 2
SUMMARY
The resistance of cancers to conventional therapies has inspired the search for novel
anti-cancer strategies. Two such promising approaches are gene therapy and
virotherapy. Gene therapy involves the delivery of therapeutic transgenes, frequently
by means of viral vectors, to tumor tissues to selectively kill them. In contrast,
virotherapy is defined as killing of cancer cells by specific virus infection, replication,
cell lysis and virus spread by so-called oncolytic viruses.
Successful implementation of gene therapy requires efficient and targeted gene
transfer vectors. Therapeutic gene transfer by replication-deficient or more recently
by replication-competent/oncolytic adenoviruses (OAds) has shown much promise.
However, for specific applications an external control of transgene expression might
be crucial. The 1st project, for the first time, investigated the feasibility of a 245bp
fragment of the human hsp70B’ promoter for heat-regulated transgene expression in
combination with either replication-deficient or oncolytic adenoviruses. In transient
transfection assays this promoter demonstrated low basal activities with induction
ratios up to 3741-fold. In context of a replication-deficient Ad, heat-stimulus resulted
in 586-fold increased luciferase activity for SK-MEL-28 cells, but was only 44-fold for
HeLa cells due to higher basal activities. To improve the heat-inducibility, two novel
Ad vectors with an insulated hsp70B’ promoter were developed that feature stringent
heat-inducible gene expression with induction ratios up to 8000-fold. However, in a
melanoma-targeted OAd regulation of the hsp70B’ promoter was lost specifically
during late replication in permissive cells and could not be restored by insulators. In
summary, this study revealed that Ad vectors with insulated hsp70B’ promoter have
potential for gene therapy applications that require external control of transgenes.
Moreover, it shows that vector replication can deregulate inserted promoters, which
needs to be considered for the development of transcriptionally regulated oncolytic
gene transfer vectors.
Adenoviruses have been intensively exploited for virotherapy approaches. Clinical
trials have demonstrated proof of principle and a favourable safety profile, but
insufficient therapeutic efficacy for these viruses. The 2nd project was designed to
increase the therapeutic potency of OAds by combining two strategies: Deletion of
the anti-apoptotic early viral gene E1B19K aimed at accelerated virus spread by early
viral release from tumor cells, and was coupled with strategies for insertion of
transgenes into the late viral transcription unit. Here the goal was the replication-
dependent expression of therapeutic genes to work in concert with viral oncolysis
(“armed” OAd). However, it remained to be demonstrated whether early lysis of
cancer cells aborts efficient transgene expression or better spread might increase it.
Deletion of E1B19K was analyzed in a panel of tumor cell cultures that resulted either
in increased early viral release and enhanced cell lysis or in reduced virus replication
and oncolysis dependent on the cell type. It could be shown that these opposing
phenotypes do not result from differences in apoptosis induction. Deletion of E1B19K
rather affected virus replication and release distinctly. Nevertheless, reporter gene
assays revealed that E1B19K deletion, despite of earlier induction of apoptosis
during viral replication, in cells susceptible for early lysis did not interfere with late
transgene expression; luciferase expression by the E1B19K- virus was even superior
over time compared to the E1B19K+ virus due to better virus spread. Thus, the
E1B19K- virus showed >3 orders of magnitude increased oncolysis and transgene
expression 10 days post-infection. Importantly, this study shows that early virus
release accelerates spread and tumor cell killing and even dramatically increases
expression of transgenes inserted into the viral genome in several tumor types.
Considering the clearance of virus by anti-viral immunity such accelerated phenotype
has the potential to improve “armed” OAds in cancer therapy.

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