Title page
CONTENTS
CHAPTER 1. INTRODUCTION 14
1-1. Introduction 15
1-2. Cancer gene therapy 16
1-3. Somatic gene therapy 17
1-4. Stem cell-based gene therapy 17
1-5. Methods for delivering therapeutic transgenes 18
1-5-1. Viral approaches to transgene delivery 18
1-5-2. Nonviral approaches to transgene delivery 20
2. Nonviral gene delivery 20
2-1. Liposome-mediated gene transfer 20
2-2. Polymer-mediated gene delivery 22
2-3. Electrophoresis 22
2-4. Particle bombardment 23
2-5. DNA injection into tissues 23
3. Targeting of gene therapy 24
3-1. Tissue-specific promoters 24
3-1-1. Prostate-specific expression 25
3-1-2. Liver-specific expression 25
3-1-3. Thyroid-specific promoters 25
3-2. Tumor specific promoters 26
3-2-1. Cancer-specific promoters 26
3-2-2. Tumor-type-specific promoters 26
CHAPTER 2. ERYTHROCYTE GHOST-MEDIATED GENE DELIVERY FOR PROLONGED AND BLOOD-TARGETED EXPRESSION 28
ABSTRACT 29
INTRODUCTION 31
METHODS 35
RESULTS 40
DISCUSSION 65
CHAPTER 3. DIFFERENTIAL EFFECTS OF PROMOTERS AND PROLONGED GENE EXPRESSION OF PLASMID DNA ADMINISTERED IN LOW MOLECULAR WEIGHT POLYETHYLENIMINE 71
ABSTRACT 72
INTRODUCTION 74
METHODS 78
RESULTS 85
DISCUSSION 116
CHAPTER 4. CONCLUSIONS 121
REFERENCES 123
Figure 1-1. Entrapment efficiency of plasmid loaded erythrocyte ghost 41
Figure 1-2. Pictures of erythrocyte ghost containing labeled fluorescent DNA 42
Figure 1-3. Blood concentration-time profiles of erythrocyte ghostmediated plasmid DNA delivery 43
Figure 1-4. Biodistribution of plasmid DNA following erythrocyte ghost-mediated delivery 45
Figure 1-5. Biodistribution of plasmid DNA following erythrocyte ghost-mediated delivery 46
Figure 1-6. Expression of plasmid DNA following erythrocyte ghost-mediated delivery 47
Figure 1-7. DNase I test for plasmidDNA 'encapsulated' in a erythrocyte ghost 48
Figure 1-8. Blood-targeted expression of plasmid DNA delivered using erythrocyte ghost 50
Figure 1-9. Expression of plasmid DNA in the subpopulation of blood cells 52
Figure 1-10. Expression of plasmid DNA following erythrocyte ghost-mediated delivery 53
Figure 1-11. Bone marrow-specific expression of plasmid DNA delivered using erythrocyte ghost 54
Figure 1-12. Expression of plasmid DNA in the subpopulation of blood cells 55
Figure 1-13. Representative data 56
Figure 1-14. Biodistribution of plasmid DNA following opsonized erythrocyte ghost-mediated delivery 58
Figure 1-15(a). Biodistribution of plasmid DNA following opsonized erythrocyte ghost-mediated delivery 59
Figure 1-15(b). Biodistribution of plasmid DNA following opsonized erythrocyte ghost-mediated delivery 60
Figure 1-16. Representative gel 61
Figure 1-17. Blood concentration-time profiles of opsonized erythrocyte ghost-mediated plasmid DNA delivery 62
Figure 1-18. Histology of the organs after repeated dosing of plasmid DNA-loaded erythrocyte ghost 64
Figure 2-1(a). Structure of expression vector pVAXmIL-2 86
Figure 2-1(b). Structure of expression vector pUBImIL-2 87
Figure 2-1(c). Structure of expression vector pEF1αmIL-2 88
Figure 2-3. The mIL-2 expression level by several types of promoters in bone marrow stromal cell 90
Figure 2-4. ThemIL-2 expression level by several types of promotersin C2C12 cell 91
Figure 2-5. ThemIL-2 expressionlevel by several types of promotersin TF-1 and bone marrow stromal cell 93
Figure 2-6. Images of GFP fluorescence in stromal cell 94
Figure 2-7. Representative gel 95
Figure 2-8(a). Organ concentration-time profiles of PEI/DNA complexes 97
Figure 2-8(b). Organ concentration-time profiles of PEI/DNA complexes 98
Figure 2-8(c). Organ concentration-time profiles of PEI/DNA complexes 99
Figure 2-8(d). Organ concentration-time profiles of PEI/DNA complexes 100
Figure 2-8(e). Organ concentration-time profiles of PEI/DNA complexes 101
Figure 2-8(f). Organ concentration-time profiles of PEI/DNA complexes 102
Figure 2-9(a). Expression level-time profiles of PEI/DNA complexes in heart 103
Figure 2-9(b). Expression level-time profiles of PEI/DNA complexes in spleen 104
Figure 2-9(c). Expression level-time profiles of PEI/DNA complexes in kidney 105
Figure 2-9(d). Expression level-time profiles of PEI/DNA complexes in liver 106
Figure 2-9(e). Expression level-time profiles of PEI/DNA complexes in lung 107
Figure 2-9(f). Expression level-time profiles of PEI/DNA complexes in ovary 108
Figure 2-10. Blood concentration-time profiles of PEI 2-kD and PEI 25- kD 110
Figure 2-11(a). Cellular toxicity of PEI/DNA complexes after repeated dosing 112
Figure 2-11(b). Cellular toxicity of PEI/DNA complexes after repeated dosing 113
Figure 2-11(c). Cellular toxicity of PEI/DNA complexes after repeated dosing 114
Figure 2-12. Representative gel for cellulart oxicity of PEI/DNA complexes after repeated dosing 115