Selective expansion of GP 91_1hnp_1hnh_1hno_1hnx gene-modified murine haematopoeitic stem cells [Elektronische Ressource] / by Linping Chen

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phoxSELECTIVE EXPANSION OF GP91 GENE-MODIFIED MURINE HAEMATOPOEITIC STEM CELLS Thesis submitted for the degree of Doctor of Nature Science Institute of Pharmaceutical Chemistry Faculty of Biochemistry, Chemistry and Pharmacy Johann Wolfgang Goethe-University of Frankfurt/Main By Linping Chen From Chengdu, P. R. China Frankfurt/Main, Germany 2005 phoxSelektive Expansion von GP91 Gen-modifizieten Murinen Hämatopoetischen Stammzellen Dissertation zur Erlangung des Doktorgrades der Naturwissenschaften vorgelegt beim Fachbereich Biochemie, Chemie und Pharmazie der Johann Wolfgang Goethe-Universität in Frankfurt am Main von Linping Chen aus Chengdu, V.R. China Frankfurt am Main 2002 DF1 Vom Fachbereich Biochemie, Chemie und Pharmazie der Johann Wolfgang Goethe-Universität als Dissertation angenommen. Dekan: Prof. Dr. Harald Schwalbe 1. Gutachter: Prof. Dr. Dieter Steinhilber 2. Prof. Dr. Winfried Wels “Scientists are painfully aware that they do not know everything but they think they can often recognize nonsense when they come across it.

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phoxSELECTIVE EXPANSION OF GP91 GENE-
MODIFIED MURINE HAEMATOPOEITIC STEM
CELLS



Thesis submitted for the degree
of Doctor of Nature Science





Institute of Pharmaceutical Chemistry
Faculty of Biochemistry, Chemistry and Pharmacy
Johann Wolfgang Goethe-University of Frankfurt/Main











By
Linping Chen
From Chengdu, P. R. China




Frankfurt/Main, Germany 2005 phoxSelektive Expansion von GP91 Gen-modifizieten
Murinen Hämatopoetischen Stammzellen



Dissertation
zur Erlangung des Doktorgrades
der Naturwissenschaften




vorgelegt beim Fachbereich Biochemie, Chemie und Pharmazie
der Johann Wolfgang Goethe-Universität
in Frankfurt am Main












von
Linping Chen
aus Chengdu, V.R. China









Frankfurt am Main 2002
DF1






























Vom Fachbereich Biochemie, Chemie und Pharmazie der Johann Wolfgang Goethe-
Universität als Dissertation angenommen.




Dekan: Prof. Dr. Harald Schwalbe
1. Gutachter: Prof. Dr. Dieter Steinhilber
2. Prof. Dr. Winfried Wels




















“Scientists are painfully aware that they do not know
everything but they think they can often recognize nonsense
when they come across it.“
- Francis Crick -

For my parents Jun Chen and Mingqing He











Table of Contents
TABLE OF CONTENTS


TITLE PAGE
1
6 ABBREVIATIONS

10 1 Introduction

1.1Haematopoiesis 10
1.1.1 Haematopoietic Stem Cells 10
1.1.2 Identification of Haematopoietic Stem Cells and Progenitor Cells 12
1.1.3 Functional Assays for Haematopoietic Stem and Progenitor Cells 15
1.1.3.1 In Vitro Assays 15
1.1.3.2 In Vivo16
1.1.4 Haematopoietic Stem Cells as Targets for Gene Therapy 17

1.2 Chronic Granulomatous Disease 19
1.2.1 Clinical Features and Diagnosis of X-linked CGD 20
1.2.2 Molecular Background of Chronic Granulomatous Disease 21
1.2.3 Treatment and Prognosis of CGD 23

1.3Gene therapy 24
1.3.1 Retroviral Gene Transfer Vector Systems 25
1.3.1.1 Gammaretroviral Vectors 27
1.3.1.2 Lentiviral Gene Transfer 30
1.3.2 CGD as Model of Gene Therapy 33
1.3.2.1 In Vitro and In vivo Experiments of Gene Therapy for CGD 33
1.3.2.2 Phase-I Clinical Trials of Gene Therapy for CGD 34
1.3.3 Drawbacks of Gene Therapy for Inherited Diseases of the Haematopoietic 35
System

1.4 Selection of Genetically Modified Haematopoietic Stem Cells 36
1.4.1 The Homeobox B4 Transcription Factor as a Candidate for Selective 37
Expansion of Gene-modified Haem Cells
1.4.2 Drug Resistance Genes as Selective Cassettes for Gene-modified 39
Haematopoietic Stem Cells
1.4.3 Direct Growth Advantage with Chimeric Receptors from Cytokine or 40
Growth Factor Receptors

1.5 Epidermal Growth Factor receptor (EGFR) and prolactin Receptor (PrlR) 42
1.5.1 al Growth Factor Receptor 43
1.5.1.1 Structure and Signalling Pathway of EGFR 43
1.5.1.2 Physiological and Patho-physiological Roles of EGFR 44
1.5.2 Prolactin and its Receptor (PrlR) 45
1.5.2.1 Structure and Signalling Pathway of PrlR 45
1 Table of Contents
1.5.2.2 Functions of Prolactin /Prlactin Receptor in haematopoesis 48

2 Goals of The Present Study 49

3 Materials and Methods 50

3.1 Materials for Molecular Biological Experiments 50
3.1.1 Equipments and Necessary Materials 50
3.1.2 Chemicals and Reagents for Cell Culture 51
3.1.3 Enzymes 53
3.1.4 Cytokines 53
3.1.5 Antibodies 53
3.1.5.1 Antibodies for Western Blot and Intracellular Staining 53
3.1.5.2 Antibodies for FACS-analyse 54
3.1.6 Reagents and Enzymes for Molecular Biological Experiments 54
3.1.7 Oligonucleotides 56
3.1.7.1 Primer for PCR 56
3.1.7.2 Oligonucleotides for Cloning 57
3.1.7.3 Oligonucleotides for Sequencing 57
3.1.7.4 Primers for Integration Test (LM-PCR) 58
3.1.8 Vectors 58
3.1.9 Bacterial E.Coli Strains 59
3.1.10 Medium for Bacterial Culture 59
3.1.10.1 5 x LB-liquid medium 59
3.1.10.2 LB-Agar 59
3.1.11 Cell lines 60

3.2 Materials for animal experiments 60
3.2.1 Mice 60
3.2.2 Materials for Preparation of Mice 60

3.3 Molecular Biological Methods 61
3.3.1 Working with E.coli and Preparation of Plasmid DNA and Genomic DNA 61
3.3.1.1 Transformation of Competent Bacterial Strains 61
3.3.1.2 Preparation of Plasmid DNA (Mini prep) 61
3.3.1.3 Plasmid Maxi-Preparation via JETstar Genomed-Columns (Combination 62
of an Anion Exchange Resin)
3.3.1.4 Preparation of Genomic DNA from Cultured Eukaryotic Cells with 62
DNeasy Kit (Qiagen)
3.3.2 Enzymatic Modification of Nucleotide Acids 63
2 Table of Contents
3.3.2.1 Restriction of Plasmid DNA 63
3.3.2.2 Dephosphorylation of Linear Plasmid-DNA by Alkaline Phosphatase CIP 63
(calf intestinal phosphatase)
3.3.2.3 Fill-in of 5’-Overhangs to Form Blunt Ends by Klenow-Reaction 64
3.3.2.4 Phenol-Chloroform Extraction for Purification of DNA Fragment 64
3.3.2.5 Ligation of DNA Fragments 64
3.3.2.6 Transformation of TOPO-Ligation Product in TOP 10F’ E.coli 65

3.4 Polymerase Chain Reaction (PCR) 65
3.4.1 PCR of Plasmid DNA for Amplification of DNA Sequences 65
3.4.2 PCR on Genomic DNA for Determination of Integration of Viral Vector 66
3.4.3 Ligation Mediated PCR (LM-PCR) 67

3.5 Working with Proteins 69
3.5.1 Preparation of Whole Cell Lysates 69
3.5.2 Bradford Protein Assay 69
3.5.3 Western Blotting 70
3.5.3.1 Discontinuous SDS-PAGE 70
3.5.3.2 Western Blotting 71
3.5.3.3 Immunostaining of Blotted Proteins 71
3.5.4 Electrophoretic Mobility Shift Assay (EMSA) 72

3.6Cell Culture 73
3.6.1 Cell Culture Conditions and Passaging of Cells 73
3.6.2 Assessing Cell Viability by Trypan Blue Exclusion 73
3.6.3 Freezing and Thawing Cells 74
3.6.4 Primary Mouse Bone Marrow Cells 74
3.6.5 Retroviral Virus Particle Production via Calcium Phosphate Mediated 74
Transfection
3.6.6 Lentiviral Virus Particle Production via Calcium76
Transfection of 293t Cells
3.6.7 Transduction of BaF/3 Cells and Primary mBM Cells with Retroviral 76
Particles
3.6.8 CFU-assay with Methylcellulose (StemCell Technologies Inc) 77
3.6.9 Selective Expansion of Transduced BaF/3 Cells and mBM Cellswith 78
Small Molecular Dimerizer AP20187

3.7Animal Experiments 80
3.7.1 Breeding of Animals 80
3.7.2 Preparation of Murine Bone Marrow Cells 80
TM 3.7.3 Enrichment of Sca1 Positive Cells from Total mBM Cells with EasySep 80
ki (SClThl)
3 Table of Contents
kit (StemCell Technology)
3.7.4 Bone Marrow Transplantation of Genetically Modified Cells 81

3.8Flow Cytometry 81

3.9Intracellular Immunostaining 82

4Results 84

phox4.1 HOXB4 Induces Selective Expansion of gp91 Gene-Modified Murine 84
HSCs
4.1.1 Cloning and Titre of Bicistronic Construct Sxgs-HOXB4 85
4.1.2 In Vitro Functional Analysis of Sxgs-HOXB4 Transduced Primary BM 86
Cells
4.1.3 In Vivo Experiments 89

4.2 Testing of Truncated Epidermal Growth Factor Receptor (EGFR) and 91
Prolactin Receptor (PrlR) as Selection Molecules
4.2.1 Cloning of Lentiviral Selection Constructs Derived from the EGF and 91
Prolactin Receptors
4.2.2 Lentiviral Vector Production and Titre Determination on 293T Cells 93
4.2.3 Selective Proliferation Advantage Supplied by S-NF2EGFR and S- 94
NF2PrlR in Transduced BaF/3 Cells
4.2.4 Reversibility of Dimerizer-Induced Selective Expansion of BaF/3 Cells 96
Expressing EGFR and PrlR Fusion Genes
4.2.5 Protein Detection of Fusion Constructs by Western Blotting 97

4.3 Development of a Bicistronic Retroviral Vector Consisting of the 98
phoxTherapeutic Gene gp91 and a Prolactin-Receptor-Based Selection
Cassette
4.3.1 Cloning of Retroviral Constructs Sxgs-NF2 and Sxgs-NF2PrlR 99
4.3.2 Production and Titre Determination of Sxgs-NF2 and Sxgs-NF2PrlR 100
Retroviral Supernatants
4.3.3 Functional Analysis of the Sxgs-NF2 and Sxgs-NF2PrlR Constructs in 101
Transduced Murine BaF/3 Cells
4.3.4 103
Transduced Murine Primary BM Cells

4.4 Development of Bicistronic Retroviral Vectors Sxgs-F2 and Sxgs-F2PrlR 105
Containing an Intracellular Signalling Molecule
4.4.1 Cloning of Constructs Sxgs-F2PrlR and Sxgs-F2 106
4.4.2 Virus Particle Production in Phoenix-E Cells and Titre Determination on 106
SC1 Cells
4.4.3 Cytoplasmic Localisation of Novel Fusion Protein F2PrlR 108
4 Table of Contents
4.4.4 Selective Expansion of Transduced BaF/3 Cells Caused by the Sxgs- 108
phoxF2PrlR Construct Containing gp91
4.4.5 The Proliferation Effect Is Reversible After Removal of the Dimerizer 112
4.4.6 Identification of Fusion Protein Expression via Western Blotting 113
4.4.7 Examination of Downstream Signal Transduction Pathways Activated by 114
the Sxgs-F2PrlR Construct
4.4.8 Selective Proliferation Advantage of Sxgs-F2PrlR-Transduced Primary 117
Sca1+ mBM Cells
4.4.9 The Selected Sxgs-F2PrlR-Transduced Cells Maintain Multilineage 121
Differentiation Ability
4.4.10 Maintenance of Early Haematopoietic Marker Gene Expression in ex vivo- 122
Cultured Cells
4.4.11 CFU Assay of ex vivo Selectively Expanded BM Cells 125
4.4.12 Polyclonal Provirus Integration of Genomic DNA in AP20187-Induced 127
Selective Expansion of Murine HSCs Transduced with Sxgs-F2PrlR
Retroviral Particles
4.4.13 Ex vivo expanded cells retain the capacity to repopulate the bone marrow 130
of lethally irradiated mice
4.4.14 Transgene Analysis of Genomic DNA from Recipient Mice Post- 131
Transplantation
4.4.15 134
4.4.16 Determination of Progenitor Cells in ex vivo-Cultured BM Cells from 136
Recipient Mice using the CFU Assay

5Discussion 139

5.1HOXB4 induced ex vivo murine haematopoietic stem cell proliferation but 139
did not confer a growth advantage in vivo
5.2 A Fusion Protein Containing the Intracellular Signalling Domain of the 141
EGF Receptor Constitutively Induced Proliferation of BaF/3 Cells
5.3 Dimerizer Induced Prolactin Receptor Signalling Promotes Expansion of 143
Murine haematopoietic Stem Cells
5.4 Perspectives 151

6Summary 153

7Zusammenfassung 155

8References 160
Acknowledgements 183

Curriculum Vitae 184

5