Analysis of adenovirus-host interactions to improve recombinant adenoviral vectors for gene therapy [Elektronische Ressource] / Christina Theresa Rauschhuber

Christina Rauschhuber

Description

Dissertation zur Erlangung des Doktorgrades der Fakultät für Chemie und Pharmazie der Ludwig-Maximilians-Universität München Analysis of Adenovirus-Host Interactions to Improve Recombinant Adenoviral Vectors for Gene Therapy Christina Theresa Rauschhuber aus Eggenfelden 2011Erklärung zur Dissertation Diese Dissertation wurde im Sinne von § 13 Abs. 3 bzw. 4 der Promotionsordnung vom 29. Januar 1998 (in der Fassung der vierten Änderungssatzung vom 26. November 2004) von Frau PD Dr. Anja Ehrhardt betreut und von Herrn Prof. Dr. Ernst Wagner von der Fakultät für Chemie und Pharmazie vertreten Ehrenwörtliche Versicherung Die vorliegende Dissertation mit dem Titel: „ Analysis of Adenovirus-Host Interactions to Improve Recombinant Adenoviral Vectors for Gene Therapy „ wurde selbstständig verfasst und alle benutzten Hilfsmittel vollständig angegeben. Die Dissertation wurde nicht schon in einer anderen Doktorarbeit verwendet. München, den 18.01.2011 Christina Rauschhuber Dissertation eingereicht am 18.01.2011 1. Gutachter: Prof. Dr. Ernst Wagner 2. Gutachterin PD Dr. Anja Ehrhardt Mündliche Prüfung am 03.03.2011 Table of contents Table of contents Zusammenfassung......................................................................................................  Abstract.....................................................................................

Informations

• Publié par: ludwig-maximilians-universitat_munchen
• Publié le: 01 janvier 2011
• Nombre de lectures: 234
• Langue: Deutsch
• Poids de l'ouvrage: 5 Mo

Extrait

nechnM ttrsievinU-Maximilians-Ludwig

2011

aus Eggenfelden

Christina Theresa Rauschhuber

Gene Therapy

Improve Recombinant Adenoviral Vectors for

Analysis of Adenovirus-Host Interactions to

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Erklrung zur Dissertation

Diese Dissertation wurde im Sinne von ¤ 13 Abs. 3 bzw. 4 der Promotionsordnung vom 29.
Januar 1998 (in der Fassung der vierten nderungssatzung vom 26. November 2004) von
Frau PD Dr. Anja Ehrhardt betreut und von Herrn Prof. Dr. Ernst Wagner von der Fakultt
fr Chemie und Pharmazie vertreten
Ehrenwrtliche Versicherung
Die vorliegende Dissertation mit dem Titel:
ã Analysis of Adenovirus-Host Interactions to Improve Recombinant Adenoviral Vectors for
Gene Therapy ã
wurde selbststndig verfasst und alle benutzten Hilfsmittel vollstndig angegeben.
Die Dissertation wurde nicht schon in einer anderen Doktorarbeit verwendet.
Mnchen, den 18.01.2011

Dissertation eingereicht am 18.01.2011
1. Gutachter: Prof. Dr. Ernst Wagner
2. Gutachterin PD Dr. Anja Ehrhardt
Mndliche Prfung am 03.03.2011

Christina Rauschhuber

Table of contents

Table of contents

Zusammenfassung......................................................................................................

Abstract........................................................................................................................

1Introduction............................................................................................................1

1.1Adenoviral Vectors...................................................................................................2
1.1.1Adenovirus taxonomy and virus particle composition...........................................2
1.1.2Infection and replication cycle of adenoviruses....................................................4
1.1.3Types of recombinant adenoviral vectors and their application in gene therapy..5
1.1.4Adenoviral vector modifications............................................................................9
1.2RNA interference.....................................................................................................13
1.2.1The RNA interference mechanism.....................................................................13
1.2.2Viral miRNAs and RNAi suppressor proteins.....................................................15
1.2.3Adenovirus associated RNAs (VA-RNAs)..........................................................17
1.3RNAi in gene therapeutic applications.................................................................18
1.4Aims of the project.................................................................................................22

2Treatment of HBV infections by shRNAs delivered by an high-capacity
adenoviral vector.................................................................................................23

2.1Materials and Methods...........................................................................................25
2.1.1DNA constructs and enzymes............................................................................25
2.1.2Cell lines and viruses..........................................................................................26
2.1.3In vitro studies to characterize efficacy of HC-AdV mediated delivery of short-
hairpin RNAs to reduce hepatitis B virus infection..............................................27
2.1.4Animal studies....................................................................................................27
2.1.5Southern Blot analysis to measure HBV genome replication.............................28
2.1.6Northern Blot to approve processing of short-hairpin RNAs...............................28
2.2Results.....................................................................................................................29
2.2.1Cloning of high-capacity adenoviral vectors.......................................................29
2.2.2Helper virus amplification...................................................................................30
2.2.3Large scale production of high-capacity adenoviral vectors in a spinner flask
system................................................................................................................30
2.2.4Purification of HC-AdV by cesium chloride centrifugation and buffer exchange32
2.2.5Titration of HC-AdV by optical density and quantitative Real-Time PCR...........34

Table of contents

2.2.6HC-AdVs for the delivery of short-hairpin RNAs to mediate inhibition of luciferase
expression by RNAi in vitro and in vivo..............................................................35
2.2.7Inhibition of hepatitis B virus infection in vitro.....................................................37
2.2.8HC-AdV results in reduction of hepatitis B surface antigen in small animal
models for hepatitis B infection...........................................................................39
2.3Discussion...............................................................................................................42

3Transcriptional activity of inverted terminal repeats of various human
adenovirus serotypes..........................................................................................45

3.1Materials and Methods...........................................................................................46
3.1.1Cell lines and viral lysates..................................................................................46
3.1.2Plasmid constructs.............................................................................................46
3.1.3Dual luciferase reporter assay to measure transcriptional activity of the ITRs...47
3.1.45«rapid amplification of cDNA ends....................................................................48
3.2Results.....................................................................................................................48
3.2.1Constructs to analyze transcriptional activity of ITRs from different adenovirus
serotypes............................................................................................................48
3.2.2In silico analysis of transcriptional binding sites of ITRs.....................................49
3.2.3Transcriptional activity of ITRs derived from different adenoviral serotypes......50
3.2.4Influence of ITR sequences upstream of the PGK promoter..............................52
3.3Discussion...............................................................................................................53

4The RNA interference pathway is responsible for silencing of transgene
expression after Sleeping Beauty mediated transposition..............................55

4.1Materials and Methods...........................................................................................56
4.1.1Plasmid construction..........................................................................................56
4.1.2Testing functionality of P19 in mammalian cells by luciferase assays...............57
4.1.3Generation of cell lines stably expressing P19...................................................58
4.1.4Western Blot analysis to investigate P19 protein expression.............................58
4.1.5P19 specific reverse transcription PCR to analyse expression from retrovirus
transduced cells..................................................................................................59
4.1.6Quantitative Real-Time PCR for quantification of HoxB8 expression levels......59
4.1.7Colony forming assays for quantification of integration events..........................59
4.2Results.....................................................................................................................60
4.2.1Analysis of the functionality of P19 in mammalian cells.....................................60
4.2.2Generation and characterization of a stable p19 expressing cell line by retrovirus
transduction and plasmid transfection................................................................62

Table of contents

4.2.3Influence of the RNA interference pathway on different integration machineries
............................................................................................................................64
4.3Discussion...............................................................................................................70
5Adenovirus 5 replication is enhanced by RNAi knockdown............................74
5.1Materials and Methods...........................................................................................76
5.1.1Plasmid constructs.............................................................................................76
5.1.2Cell lines and viruses..........................................................................................80
5.1.3Analysis of CAR expression of HEK293 and B6 cells........................................81
5.1.4Isolation of wtAd DNA from purified particles.....................................................81
5.1.5Analysis of adenovirus replication in HEK293 and B6 cells...............................82
5.1.6RNA isolation and rev