RNA Technologies and Their Applications (eBook)

RNA Technologies and Their Applications 3
Preface 5
Contents 9
Contributors 11
The Key Features of RNA Silencing 17
1 Introduction 18
2 RNA Silencing Effector as a Two-Component System 21
3 Small RNA Biogenesis 22
3.1 miRNAs and siRNAs 22
3.2 Dicer-Independent Pathways 24
3.2.1 piRNAs 24
3.2.2 Secondary siRNAs in Worm 26
3.3 Endo-siRNAs 27
4 RISC Loading, Sorting, and Target-Sensing of Small RNAs 28
4.1 Sorting by Precursor Structures 30
4.2 Sorting by the 5 Ends 31
4.3 Sorting by Dicer Processing Polarity 31
4.4 Target-Sensing 32
5 Safeguards for RNA Silencing Pathways 33
6 Effector Modes of RNA Silencing Pathways 34
7 Regulations of RNA Silencing Pathways 35
7.1 Processing 35
7.2 Modification 37
7.3 RISC Activity 37
8 Perspective 38
References 39
Selected Strategies for the Delivery of siRNA In Vitro and In Vivo 45
1 Introduction 47
2 Mechanism of RNA Interference 48
3 Naked Delivery of siRNA In Vitro 48
3.1 Cellular Uptake of Naked Nucleic Acids 48
3.2 The Phosphorothioate-Stimulated Cellular Delivery of siRNA 50
3.3 The siRNA-Peptide Conjugate Approach 51
3.4 Intracellular Release of siRNA: A Major Hurdle 54
4 CPP-Mediated siRNA Delivery 55
4.1 Cell-Penetrating Peptides 55
4.2 Selected Examples of CPP-Mediated siRNA Delivery 57
5 Selected Examples of siRNA Delivery In Vivo 63
6 Conclusions and Future Prospects 66
References 67
RNAi Suppression and Its Application 75
1 RNA Interference 77
2 RNAi-Directed Viral Immunity 81
3 Identification and Function Characterization of Viral RNAi Suppressors 84
3.1 Agrobacterium-Mediated Transient Suppression Assay 85
3.2 Reversal of Transgene-Induced Gene Silencing 86
3.3 Grafting Assay 86
3.4 Replication Rescue of Mutant Viruses Defective in RNAi Suppression 86
4 Function Mechanism of Viral RNAi Suppressors 88
4.1 Viral Suppressors that Bind Viral dsRNA to Interfere with viRNA Biogenesis 89
4.2 Viral Suppressors that Target Virus-Derived siRNA for RNAi Suppression 91
4.3 Viral Suppressors that Target RNAi Effectors for Suppression 93
4.4 Viral Suppressors that Suppress Systemic RNAi 95
5 RNAi Suppressors of Nonviral Origin 96
5.1 Suppression of RNAi by Bacterial Pathogens 96
5.2 Cellular RNAi Suppressors 98
6 Biotechnological Application of RNAi Suppressors 99
6.1 Enhance Gene Expression for Rapid Function Analysis and Mass Production of Valuable Protein 99
6.2 Serve as Molecular Tools to Probe Various RNAi-Directed Functions 100
Appendix 101
References 103
Strategies to Prevent siRNA-Triggered Cellular Toxicity 109
1 Introduction 110
2 Cellular Sensors of siRNA-Triggered Innate Immune Response 112
2.1 TLR-Mediated Innate Immunity 113
2.2 Non-TLR-Mediated Innate Immunity 113
3 Cellular Sequels After siRNA-Triggered Innate Immune System Activation 114
4 Overcoming Synthetic siRNA-Triggered Innate Immune Response 115
5 Overcoming shRNA-Triggered Innate Immune Response 116
6 shRNA-Mediated Disruption of the Endogenous miRNA Machinery 118
7 Conclusions 119
References 119
RNAi in Malignant Brain Tumors: Relevance to Molecular and Translational Research 123
1 Introduction 124
1.1 Diagnostic Characteristics of Diffuse Astrocytic Tumor 124
1.2 Clinical Course 125
1.2.1 Symptoms 125
1.2.2 Prognosis 126
1.3 Obstacles in Treatment of Diffuse Astrocytic Tumors 126
2 RNAi for Glioma: from Bench to Clinic 127
2.1 Preclinical Studies 128
2.2 Target Genes for Silencing 129
2.3 Problems in Clinical Translation 130
2.3.1 Specificity 131
Off-Target Effect 131
Interferon Response 131
2.3.2 Instability 132
2.3.3 Delivery 132
Systemic Route 133
Local Route 134
3 miRNAs in Glioma 135
3.1 Molecular Pathology of Aberrant miRNAs in Glioblastoma 136
3.1.1 Upregulated miRNAs 136
miR-21 136
miR-26a 136
miR-125b 136
miR-221-222 Cluster 138
miR-296 138
3.1.2 Downregulated miRNAs 138
miR-7 138
miR-15 139
miR-124 and miR-137 139
miR-128 139
miR-146b 139
miR-181 Family 140
4 Conclusions and Perspective 140
References 140
Silencing Huntington´s Disease Gene with RNAi 146
1 Introduction 147
2 A New Approach to Understanding Normal Function of Wild-Type Huntingtin 148
3 Nonallele-Specific Silencing of Huntingtin 150
4 Allele-Specific Silencing of Mutant Huntingtin 159
5 Current Challenges and Future Perspectives 163
References 170
Application of Dicer-Substrate siRNA in Pain Research 176
1 RNAi in Pain Research 177
2 Potential Applications of RNA Interference for Pain Treatment 178
2.1 Ion Channels as Therapeutic Targets for Pain 178
2.2 G-Protein-Coupled Receptors as Pain Targets 181
3 Advantages of DsiRNA Over Conventional siRNA 182
3.1 Inherent Character of DsiRNA 183
3.1.1 Dodging Nucleases Attack 183
3.1.2 Keeping ``On-Target´´ 184
3.1.3 Eluding the Unpredictable 185
3.1.4 Sharing Is Not Always Best 185
3.2 Molecular Mechanism of Action 186
3.2.1 Initiation of RNAi Pathways: Role of DICER 186
3.2.2 Formation of RISC Loading Complex and Its Activation 187
3.2.3 Target Recognition by RISC and Gene Silencing 188
4 DsiRNA for Efficient Silencing In Vitro 189
4.1 Methodology 189
4.1.1 Design Rules 189
4.1.2 Cell Lineage Selection 190
4.1.3 Extensive Formulation Screening 190
4.1.4 Control Your Assay 191
4.2 Validation of Knockdown Efficiency 191
4.3 Targets In Vitro 192
5 DsiRNA In Vivo: One Step Forward 195
5.1 Working Evidence of DsiRNA In Vivo 195
5.1.1 Peripheral Organs 195
5.1.2 Central Organs 196
5.2 Methodology 196
5.2.1 Formulation 196
5.2.2 Uptake Validation 197
5.2.3 Molecular Silencing Confirmation 197
5.2.4 Assessing Specificity 198
5.2.5 Pain Assessment 199
6 Conclusion and Perspectives 201
References 203
RNAi Treatment of HIV-1 Infection 206
1 The RNAi Pathway for Expression of Therapeutic siRNAs 207
2 RNAi Gene Therapy Against HIV-1 209
3 Finding the Optimal Viral Target Sites 211
4 Combinatorial RNAi 212
5 Targeting Cellular Cofactors of HIV-1 Replication 212
6 Alternative Inhibitory RNA Molecules 213
7 Preclinical Test Systems 214
8 Sequence-Specificity of RNAi Action 214
9 Safety Issues Raised in Clinical Trials 215
10 Clinical Trials 216
11 Conclusion 216
References 217
Application of RNA Interference to Treat Conditions Associated with Dysregulation of Transient Receptor Potential Vanilloid 1 224
1 TRPV1 Ion Channels 226
2 Neuropathic Pain 227
2.1 Current Treatment 227
2.2 TRPV1 and Neuropathic Pain 227
2.3 Diabetic Peripheral Neuropathy 228
3 Drug-Induced Hearing Loss 229
3.1 TRPV1 and Cisplatin Ototoxicity 229
3.2 Utility of RNAi in Treating Cisplatin Ototoxicity 230
4 Other Potential Uses of RNAi Targeting TRPV1 232
4.1 Inflammation 233
4.2 Arthritis 233
4.3 Cystitis and Bladder Hyperactivity 234
4.4 Cancer Pain 235
4.5 Obesity 236
5 Conclusions 237
References 238
Harnessing RNAi-Based Functional Genomics to Unravel the Molecular Complexity Underlying Skin Pigment Variation 242
1 Melanin: A Ubiquitous Pigment with a Multitude of Functions 243
2 Melanogenesis: Insights Uncovered by the Detailed Analysis of Genetic Disorders of Pigmentation 245
3 An RNAi-Based Functional Genomics Approach to Identify Novel Regulators of Melanogenesis in Human Cells 248
4 Integration of Multiple Systems-Level Approaches to Uncover Additional Regulators of Melanogenesis in Our Functional Genomic. 252
5 Identification of Novel Pathways that Regulate the Transcription of Melanogenic Enzymes 256
6 Identification of Novel Pathways that Regulate Melanosome Biogenesis 259
7 Identification of Regulators of Human Pigment Variation 262
8 Identification of Pharmacologic Agents that Impact Melanin Accumulation 263
9 Concluding Remarks 264
References 264
mRNA Structure and its Effects on Posttranscriptional Gene Silencing 269
1 Introduction 270
2 A Structured Target Site Reduces AON and siRNA Activity In Vitro 271
3 Analysis of Binding Affinity to mRNA and Rate Dependencies on Concentration for AON and siRNA Activity 272
4 AON and siRNA Guide Strand Have Equal Affinity for the Target mRNA 275
5 AON and siRNA Display Apparent First and Zero Order Kinetics 275
6 For Full In Vitro Activity, siRNA Require Greater Target Site Accessibility Than AON 277
7 A Double-Stranded Target Site Greatly Reduces In Vitro PTGS Activity 279
8 An AON That is More Effective Than the siRNA Against an Identical Target In Vitro is Less Effective Against the Same Target . 279
9 Discussion 282
10 Conclusions 286
References 287
Antisense RNA-Mediated Regulation of the p53 Tumor Suppressor 290
1 Antisense RNAs as Regulators of Gene Expression 291
2 Regulation of p53 at the mRNA Level 292
3 Wrap53 293
4 Future Perspectives 296
References 296
Antisense Oligonucleotides: Insights from Preclinical Studies and Clinical Trials 298
1 Introduction 300
2 Antisense Oligonucleotides in Cancer Treatment 302
2.1 BCL2 (B-cell CLL/lymphoma 2) 302
2.2 XIAP (X-Linked Inhibitor of Apoptosis) 304
2.3 Survivin, BIRC5 (Baculoviral IAP Repeat-Containing 5) 304
2.4 CLU (Clusterin) 305
2.5 TGFB2 (Transforming Growth Factor, beta 2) 306
3 Application of Antisense Oligonucleotides in Noncancerous Diseases 307
3.1 Asthma 307
3.2 Cardiovascular Disease 307
3.3 Duchenne Muscular Dystrophy - Exon-Skipping Therapy 308
3.4 Virus Infections 309
4 Specificity of Antisense-Mediated Gene Silencing 310
5 Conclusion 312
References 313
What can the New Hammerhead Ribozyme Structures Teach us About Design? 317
1 Introduction to the Hammerhead Ribozyme 318
1.1 The Genomic Ribozymes 319
1.2 What is a Hammerhead Ribozyme 319
1.3 Minimal and Full-Length Hammerhead Ribozymes 319
1.4 Expanding Biological Context 322
2 Hammerhead Ribozyme Structures 323
2.1 Three-Dimensional Structure of Minimal Hammerhead Ribozymes 324
2.2 Three-Dimensional Structures of Full-Length Hammerhead Ribozymes 325
2.2.1 Schistosomal Hammerhead Structure 325
2.2.2 Satellite Viral Hammerhead Ribozyme Structure 327
3 Structure and Mechanism 329
3.1 Acid-Base Catalysis 329
3.2 Metal Ions? 330
3.3 Substrate Binding and Specificity 331
4 Hammerhead Structure, Function, and Design 332
4.1 Minimal Hammerheads 332
4.2 Full-Length Hammerheads 332
References 334
microRNA Biogenesis and its Impact on RNA Interference 336
1 The microRNA Biogenesis Pathway 338
1.1 microRNA Gene Transcription 338
1.2 microRNA Editing: Small Changes Affect Many Steps 338
1.3 pri-miRNA Cleavage by the Microprocessor Complex 339
1.3.1 Regulation of the Microprocessor 340
1.3.2 Primary miRNA Generation in Plants 342
1.4 Nuclear Export of the microRNA Precursors by Exportin-5 343
1.5 The RISC Loading Complex 344
1.6 Terminal Loop Removal by Dicer 345
1.6.1 Control Mechanisms of the Dicer Cleavage 346
1.7 Ago2 Jumps the Queue: Generation of the ac-pre-miRNA 346
1.8 miRNA Duplex Unwinding 347
1.9 Strand Selection: Who Becomes the Guide? 348
1.10 Mediators of RNA Silencing: The Argonaute Proteins 348
1.10.1 Regulation of Ago Activity and Ago-Mediated Silencing Mechanisms 350
1.11 Half-Life and Degradation of microRNA 351
2 Implication for RNAi Technology 351
2.1 Potentials and Challenges of siRNAs as a Tool 353
2.2 siRNA Versus shRNA 354
2.3 shRNA-miR Library: Transferring microRNA Structures to Synthetic shRNAs 354
2.4 Enhancement of RNAi by microRNA Biogenesis Factors 355
2.5 microRNA Biogenesis in Health and Disease: Basis for RNAi Therapy 357
2.6 Concluding Remarks 358
References 358
MicroRNAs in Epithelial Antimicrobial Immunity 366
1 Introduction 367
2 Abundant Expression of miRNAs in Epithelial Cells 368
3 Regulation of miRNA Expression in Epithelial Cells 369
4 MicroRNAs in the Regulation of Epithelial Antimicrobial Defense 371
4.1 MicroRNAs and Maintenance of Epithelial Barrier Integrity 371
4.2 MicroRNAs and Regulation of Epithelial Intracellular Signaling Pathways 372
4.3 MicroRNAs and Expression of B7-Costimulatory Molecules in Epithelial Cells 374
4.4 MicroRNAs in the Exosomes Released from Epithelial Cells 374
4.5 MicroRNAs-Mediated Antivirus Response in Epithelial Cells 374
5 Conclusion and Perspectives 375
References 376
Emerging Roles of Long Noncoding RNAs in Gene Expression and Intracellular Organization 379
1 Introduction 380
2 Intracellular Behaviors of ncRNAs Distinct from Those of mRNAs 380
3 Unique Pathways for Long ncRNA Biogenesis 382
4 ncRNA Functions in the Regulation of Gene Expression 384
4.1 Regulation of Transcription Factor Activity by Long ncRNAs 384
4.2 ncRNA Transcription Affects Adjacent Gene Expression 386
4.3 ncRNA Recruits or Modulates Epigenetic Factors on the Chromosome 388
4.4 ncRNAs Regulate Gene Expression at Posttranscriptional Steps 390
5 Structural Roles of ncRNAs 391
6 ncRNAs in Biomedical Research 394
7 Future Directions for ncRNA Research 394
References 396
Noncoding RNAs as Therapeutic Targets 402
1 RNA-Dependent Regulation of Gene Expression 403
1.1 Gene Regulation Through Epigenetic Mechanisms 404
1.2 Controlling Transcription Machinery Activity 406
1.3 Posttranscriptional Regulatory Mechanisms 408
2 The Medical Perspective: Noncoding RNAs in Human Diseases 410
2.1 MicroRNAs 410
2.2 mRNA-Like ncRNAs 413
2.3 Other Transcripts 415
3 NcRNA-Based Therapeutic Strategies 416
References 418
Noncoding RNAs at H19/IGF2 Locus: Role in Imprinting, Gene Expression, and Associated Pathologies 428
1 The H19/IGF2 Locus and the Parental Imprinting Model 429
1.1 Overview and Description of the 11p15.5 Locus 429
1.2 The Insulator Model of Imprinting 433
1.2.1 DNA Methylation of H19 and IGF2 Genes 433
1.2.2 Histone Modifications at the H19/IGF2 Locus 434
1.2.3 The ICR or Imprinting Control Region 434
1.2.4 Imprinting and Parental Specific Chromatin Loops 436
2 The mRNA-Like Noncoding RNA H19 437
2.1 Properties and Expression 438
2.2 Functions 439
2.3 Regulation 439
3 The Noncoding Antisense RNA 91H 440
3.1 Characterization 440
3.2 Hypothesis About 91H Mechanism of Action 441
4 H19/IGF2 Locus-Associated Pathologies 442
4.1 Hormone-Dependent Cancers (Breast, Uterus) 442
4.2 Children Syndromes 444
5 Conclusion 445
References 445
Index 453