Non-viral Gene Therapy (eBook)

Foreword 5
Preface 7
Contents 10
1. Molecules for Gene Therapy 13
Recent Progress in Non-viral Gene Delivery 14
1 Introduction 14
2 Delivery of Naked DNA by Physical Methods 14
3 Cationic Polymer-Based Non-viral Vectors 16
4 Lipid-Based Non-viral Vectors 17
5 Toxicity of Non-viral Vectors 18
References 18
Physical Chemistry of DNA-Carrier Complexes 22
1 Ordered and Disordered Structures in Compact DNA 22
References 28
Liposomes 30
1 Liposomal Drug Delivery Systems for Gene Delivery In Vivo 30
2 Targeting to a Less Accessible Site (Solid Tumor) 36
3 Liposomal Gene Delivery Systems 39
References 42
Polymeric Gene Carriers 46
1 Introduction 46
2 Various Polymeric Carriers 47
3 Transfection Protocol Using Polyplexes 50
4 Biological Barriers 51
5 Conclusions 56
References 56
Development of a Supramolecular Nanocarrier for Gene Delivery Based on Cationic Block Copolymers 62
1 Introduction 62
2 Preparation of Block Copolymers as Gene Carriers 63
3 Polylysine-Based Non-viral Gene Delivery Systems 66
4 Environmentally Responsive Polycation-Based Non- viral Gene Carriers 67
5 Conclusions 70
References 70
Chitosan 74
1 Introduction 74
2 DNA/Chitosan Complexes 74
3 Chitosan Derivatives 78
4 Mechanism of Transcellular and Intracellular Transport of DNA/ Chitosan Complexes 80
5 DNA/Chitosan/Sugar-PEG-C Ternary Complexes 82
6 Conclusion 83
References 83
Dendrimers as DNA Carriers 86
1 Introduction 86
2 Transfection Ef.ciency 87
3 Factors Affecting Transfection Activity 89
4 Combining Dendrimers with Other Strategies 91
5 Dendrimer Conjugates 92
6 Conclusion 92
References 94
Use of Synthetic Peptides for Non-viral Gene Delivery 98
1 Introduction 98
2 Peptides as Cationic Moieties of Gene Carriers 98
3 Peptides as Ligands 104
4 Peptides as Tools for Endosome Disruption 106
5 Peptides as Signals for Transport to the Nucleus 108
6 Peptides as Sensors for Functional Gene Delivery 108
7 Prospects 110
References 110
An Oligonucleotide Carrier Based on b- 1,3- Glucans 114
1 Oligonucleotide/ b- 1,3- Glucan Complexes 114
2 Biofunctional Oligonucleotides, Antisense and CpG Oligonucleotides 115
3 Strategy and Chemistry 117
4 Delivery of CpG Motifs 119
5 Delivery of Antisense Oligonucleotides to Avoid Lysosomal Degradation 121
6 Conclusions 126
References 126
Biological and Chemical Hybrid Vectors 129
1 Introduction: Concept of the Hybrid Vector 129
2 Fusogenic Liposome: A Delivery Vehicle Mimicking Envelope Viruses 131
3 Peptide-Displaying Phage Particles: Delivery Vehicles Mimicking Non- envelope Viruses 134
4 Construction of Stable Replicons 137
5 Future Perspectives: Towards the Development of Ideal Hybrid Vectors 139
References 140
2. Controlled Gene Delivery 144
Pharmacokinetics of Gene Delivery in Cells 145
1 Introduction 145
2 A New Packaging Concept for Non-viral Vectors for Ef . cient Gene Delivery: Programmed Packaging 147
3 Control of the Intracellular Traf.cking of Genes 148
4 Optimization of Intracellular Traf.cking: Pharmacokinetic Considerations 156
5 Conclusions 159
References 160
Active DNA Release from Complexes 165
1 Introduction 165
2 pH-Responsive Vectors 167
3 Redox-Responsive Vectors 168
4 Photo-Responsive Vectors 170
5 Conclusions 171
References 172
Controlled Release of DNA Using Thermoresponsive Polymers 175
1 Signi.cance of DNA Release from Synthetic Gene Carriers 175
2 Stimuli-Responsive Synthetic DNA Carriers 176
3 Temperature-Responsive Polymers and Their Applications 178
4 DNA Carrier Systems Using Temperature-Responsive Polymers: Temperature- Responsive Polymeric Vector Systems 179
References 184
Active Transport of Exogenous Genes into the Nucleus 186
1 Introduction 186
2 Nuclear-Pore Complex and Nuclear Import of Karyophilic Proteins 187
3 Nuclear Import of Virus Genomes 189
4 Nuclear Import of DNA Conjugated with NLS Peptides 189
5 Nuclear Import of DNA Conjugated with DNA- Binding Proteins 191
6 Nuclear Import of DNA Conjugated with Viral Proteins 192
7 Nuclear Import of DNA Directly Conjugated with Importins 192
8 Nuclear Import of DNA with a Glycosylated Carrier 192
9 Conclusions 193
References 193
Controlled Intracellular Localization of Oligonucleotides by Chemical Conjugation 197
1 Introduction 197
2 Synthesis of Oligonucleotide-Peptide Conjugates by Solid- Phase Fragment Condensation 197
3 Nuclear and Cytoplasmic Localization of Oligonucleotide- Peptide Conjugates 199
4 Mechanisms of Cellular Uptake of Oligonucleotide- Peptide Conjugates 204
References 205
Functional Nucleotide Sequences Capable of Promoting Non- viral Genetic Transfer 208
1 Introduction 208
2 Plasmid Vectors as an Important Component of Non- viral Gene Delivery Systems 208
3 Plasmid Vector Sequence Crucially Affects the Ef.cacy of Non- viral Gene Transfer 209
4 Epstein-Barr Virus-Based Plasmid Vectors 213
5 The Sleeping Beauty Transposable Element 214
6 Conclusions 216
References 216
Shielding of Cationic Charge of the DNA Complex to Avoid Nonspeci . c Interactions for In Vivo Gene Delivery 221
1 Introduction 221
2 Interaction of DNA Complexes with Blood Components or Extracellular Matrices 222
3 Size Control of DNA Complexes 228
4 Transcription-Activating Effect of Polyanions and Polyampholytes: Ionized PEGs as Arti . cial HMG Proteins 231
References 233
In Vivo Gene Transfer by Ligand- Modi . ed Gene Carriers 236
1 Introduction 236
2 Biodistribution Characteristics of Naked pDNA 237
3 Biodistribution Characteristics of pDNA Complexed with Cationic Liposomes 238
4 Biological Barriers to Gene Delivery 238
5 In Vivo Receptor-Mediated Gene Delivery 240
References 244
Optimizing Polyplexes into Synthetic Viruses for Tumor- Targeted Gene Therapy 247
1 Introduction 247
2 Shielded Ligand-PEI Polyplexes for Tumor-Targeted Gene Transfer 248
3 Development of Novel Polymers as DNA Carriers 249
Shielding from non- specific interaction 250
4 Bioresponsive Elements for Optimizing Polyplexes into Synthetic Viruses 252
5 Conclusions 253
References 253
Gene Transfer and Target Diseases 256
1 Gene Transfer and Target Diseases 256
2 Cardiovascular Diseases 256
3 Cancer 261
4 Conclusions 262
References 263
Clinical Trials Using Non-viral Gene Delivery Systems 271
1 Introduction 271
2 Clinical Trials Using Cationic Lipid Gene Transfer 272
References 298
3. Current Protocols of Gene Delivery 301
Evaluation of Size and Zeta Potential of DNA/ Carrier Complexes 302
1 Introduction 302
2 Materials 303
3 Methods 303
4 Results 305
References 308
Observation of DNA/Carrier Complexes Under Fluorescence Microscopy 309
1 Introduction 309
2 Principle of Single Molecular Observation by Fluorescence Microscopy 310
3 Fluorescent Dyes for Observing DNA Molecules 311
4 Fluorescence Microscopy and the Recording System 311
5 Sample Preparation 312
6 Measurement of DNA Size in Solution 312
7 Observation of Each Component of the DNA Polyion Complex 313
8 Observation of the DNA Interaction with Cells 313
9 Attachment of the DNA Complexes onto Charged or Protein- Immobilized Beads 314
References 315
Synthesis of Chemically Modi.ed Chitosan and a Study of Its Gene Transfection Ef . ciency 316
1 Introduction 316
2 Materials 318
3 Methods 318
References 323
Newly Designed DNA Fragments for Gene Correction 324
1 Introduction 324
2 Materials 325
3 Methods 326
References 330
Evaluation of Gene Expression In Vivo After Intravenous and Intraportal Administration of Lipoplexes 332
1 Introduction 332
2 Materials 333
3 Methods 334
References 338
Evaluation by Southern Blot Hybridization of DNA Administered with a Gene Carrier to Organs 340
1 Introduction 340
2 Materials 341
3 Methods 341
4 Biodistribution and Pharmacokinetics of Intravenously Injected DNA with Gene Carriers 346
References 347
Evaluation of the Immune Response After Administration of Plasmid DNA- Non- viral Vector Complexes 348
1 Introduction 348
2 Materials and Methods 349
3 Comments 353
References 354
Gateway RNAi 357
1 Introduction 357
2 Gateway 358
3 Experimental Protocols 360
4 Application of Gateway System 360
5 Conclusions 365
References 368
4. Design of Genes Based on Current RNA Technology 370
Design of Intracellularly Active Ribozymes and siRNAs 371
1 Ribozymes 371
2 siRNAs, siRNA-Expression Vectors, and siRNA- Expression Library 388
3 Concluding Remarks 397
References 398
RNAi-Based Inhibition Speci.c for Mutant Alleles in Autosomal Dominant Diseases: Sequence- Dependent and - Independent Discrimination of Mutant and Wild- Type Alleles by siRNA 406
1 Introduction 406
2 Optimal Location of a Mutation for Discriminating Among Point Mutations in Familial ALS 406
3 siRNA Speci.c for G93A SOD1 with Point Mutation in Familial ALS 407
4 siRNA Speci.c for the Mutant Allele in Machado- Joseph Disease Using a Polymorphorism Related to CAG Repeat Length 408
5 Sequence-Independent Discriminations of Mutant and Wild- Type Alleles by siRNA 410
6 Discussion 410
References 412
In Vivo RNA Interference: Another Tool in the Box? 413
1 Introduction 413
2 First In Vivo RNAi Proof-of-Concept Experiment: Inoculation of siRNA 414
3 Transgenic: Random Integration of shRNA- Expressing Constructs 416
4 Transgenic Models: Targeted Insertion Approach 419
5 Future Development of In Vivo RNAi 420
6 Conclusion 426
References 427
Suppression of Gene Expression via Chromatin Remodeling and the siRNAInduced Silencing of Transcription 431
1 Epigenetics 431
2 Post-transcriptional Gene Silencing 432
3 Transcriptional Gene Silencing 433
4 RNAi-Induced Methylation of DNA in Human Cells 436
5 Chromatin-Remodeling Complexes and RNAi 436
6 Conservation of siRNA-Induced TGS and Methylation in Various Species 439
7 siRNA-Directed Methylation of the Genome 440
8 Concluding Remarks 443
References 443
Intracellular Delivery of Nucleic Acids: Differences Between Transfection and siFection Re . ect Differences Between DNA and RNA, and Between Oligodeoxynucleotides and Oligonucleotides 449
1 Introduction 449
2 Delivery of Short, Double-Stranded Synthetic RNA — siFection 452
3 Discussion 454
4 Summary 457
References 458
In Vivo Antitumor Activity of a New Cationic Liposome siRNA Complex 464
1 Introduction 464
2 A New Cationic Liposome, LIC-101 465
3 Composition and Formulation of LIC-101 465
4 Intracellular Delivery of Liposome/Nucleic Acid Complexes 466
5 Biological Activity of siRNA 467
6 Discussion 471
7 Concluding Remarks 471
References 472
Enhancing RNAi with Synthetic RNA Duplexes 473
1 Introduction to siRNA-Mediated Silencing 473
2 Characterization of siRNAs Produced from 27-mers 476
3 Testing 27-mers Against Endogenous Targets 479
4 Testing for Interferon Pathway Activation and Off- Target Effects of 27- mers 480
5 Delivery of Synthetic RNAs 482
References 482
Subject Index 484