Table of Contents
The Outlook for RNAi
Executive Summary 12
Current RNA technologies 12
Design, production and delivery of RNAi 13
The future of RNAi in research and drug discovery 14
The future of RNAi drug therapies 15
Emerging RNA technologies and future trends 16
Patents and strategic alliances in RNAi technology 17
RNAi markets and trends 18
Chapter 1 Current RNA technologies 20
Summary 20
Introduction 21
History of RNAi 21
From DNA to RNA to proteins 22
mRNA regulation 23
Gene expression 24
Antisense technology 25
Oligonucleotides (OGNs) 26
Peptide nuclei acids (PNAs) 28
Locked nucleic acids (LNA) 28
Triple helix DNA or triple helix-forming oligonucleotides (TFOs) 30
Ribozymes 31
DNAzymes 32
Aptamers 33
RNA interference 33
siRNAs versus dsRNA 36
siRNAs versus shRNA 36
Conclusions 38
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Chapter 2 Design, production and delivery
of RNAi 42
Summary 42
Introduction 43
Cost-effective RNA design 43
Cost-effective synthesis of siRNA 45
Chemical synthesis 47
Conclusions 49
In vitro transcription 50
DICER reaction 51
Expression vectors 52
DNA-directed RNAi (ddRNAi) 53
Expressed interfering RNA (eiRNA) 55
Conclusions 56
Improvements in siRNA stability 57
Chemical modifications 57
Formulation modifications 59
Small molecule conjugation 59
Synthetic vector systems 61
Conclusion 62
RNAi delivery options 63
Viral vectors 65
Conclusions 66
Chapter 3 The future of RNAi in research
and drug discovery 70
Summary 70
Introduction 71
Applications of RNAi in research 72
Functional genomics 72
Signaling pathways 75
Applications of RNAi in drug discovery 76
Gene expressions studies 76
Target validation 81
Toxicogenomics 83
Applications of RNAi in drug development 85
Transgenics 86
The impact of RNAi in R&D 92
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Chapter 4 The future of RNAi drug
therapies 96
Summary 96
Introduction 97
Shift from antisense to RNAi 98
Ocular diseases 101
Age-related Macular Degeneration (AMD) 101
Key RNAi players 103
Diabetic Retinopathy (DR) 106
Key RNAi players 106
Conclusions 107
Infectious diseases 107
Hepatitis C virus (HCV) 108
Key RNAi players 109
HIV 111
CMV (cytomegalovirus) 112
Key RNAi players 113
Conclusions 113
Respiratory 114
Respiratory Syncytial Virus (RSV) 114
Key RNAi players 114
Asthma 115
Key RNAi players 116
Cystic fibrosis 116
Key RNAi players 116
Conclusions 117
Neurological diseases 118
Huntingdon’s disease (HD) 119
Key RNAi players 120
Amyotrophic Lateral Sclerosis (ALS or Lou Gehrig's disease) 120
Key RNAi players 122
Spinal Cord Injury (SCI) 122
Key RNAi players 123
Parkinson's disease (PD) 123
Alzheimer's disease (AD) 124
Pain 125
Conclusions 126
Oncology 127
Angiogenesis 128
Key RNAi players 128
Oncogenes 129
Key RNAi players 129
Drug resistance and enhancement 131
Key RNAi players 132
Conclusions on RNAi in oncology 132
Cardiovascular diseases 132
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Key RNAi players 133
Conclusions 134
Metabolic disorders 134
Diabetes 134
Key RNAi players 135
The future role of RNAi-based therapeutics 138
Chapter 5 Emerging RNA technologies and
future trends 140
Summary 140
Introduction 140
Second generation siRNAs 142
Multifunctional siRNAs 143
Hyperfunctional or superactive siRNAs 143
No-ribose small inhibitory nucleic acids (siNAs) 145
siRNAs conjugated with small molecule drugs 145
Alternative RNA based therapies: 146
Micro RNAs (miRNAs) 146
miRNA processing 147
miRNA in embryonic development 148
miRNA in neurological disorders 149
miRNA in cancer 149
Future direction of miRNA research 149
Small nucleolar RNAs (snoRNAs) 150
Aptamers 151
Chapter 6 Patents and strategic alliances in
RNAi technology 154
Summary 154
Introduction 155
Patents for siRNA reagents 156
Patents for siRNA therapeutics 158
Alnylam Pharmaceuticals (Cambridge, MA, US) 160
Patent position 160
Strategic alliances, 2003-2005 161
Benitec Ltd (Queensland, Australia) 163
Patent position 163
Strategic alliances, 2003-2005 164
Sirna Therapeutics (formerly Ribozyme Pharmaceuticals) 165
Patent position 165
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Strategic alliances, 2003-2005 168
Acuity Pharmaceuticals (Philadelphia, PA, US) 168
Patent position 168
Strategic alliances, 2003-2005 169
Atugen AG (Dresden, Germany) 169
Patent position 169
Strategic alliances, 2003-2005 169
CytRx Labs (Los Angeles, MA, USA) 170
Patent position 171
Strategic alliances, 2003-2005 171
Intradigm (Rockville, MD, USA) 172
Nucleonics Inc. (Horsham, PA, USA) 172
Future impact of IP on RNAi research 173
Chapter 7 RNAi markets and trends 176
Summary 176
Introduction 177
The RNAi market 178
Market size and future trends 180
siRNA synthesis and delivery 182
RNAi reagents 183
RNAi in drug discovery and target validation 184
RNAi therapeutics 185
Chapter 8 Appendix 190
Acknowledgements 190
Index 191
Bibliography 193
Glossary 201
References 205
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List of Figures
Figure 1.1: History of RNAi 22
Figure 1.2: Schematic of DNA, genes and proteins 23
Figure 1.3: Schematic of gene splicing 24
Figure 1.4: Major mechanisms for antisense OGN action 25
Figure 1.5: Mechanism of preventing translation using OGN technology 27
Figure 1.6: Chemical structure of PNA versus DNA 28
Figure 1.7: Chemical structure of LNA versus RNA 29
Figure 1.8: Mechanism of preventing translation using triple helix DNA technology 30
Figure 1.9: Mechanism of preventing translation using ribozymes 31
Figure 1.10: Schematic of the mechanism of gene silencing by RNAi 34
Figure 1.11: Schematic of the mechanism of shRNAs 36
Figure 2.12: Advantages and disadvantages of siRNA synthesis methods 46
Figure 2.13: In vitro transcription of siRNAs 50
Figure 2.14: DICER digestion of dsRNAs 52
Figure 2.15: psiRNA plasmid vector system 53
Figure 2.16: Mechanism of ddRNAi 54
Figure 2.17: Chemical modifications of siRNAs increase stability and PK 57
Figure 2.18: Chol- siRNAs improve tissue uptake and PK 60
Figure 2.19: Intradigm's nano-delivery technology TargeTran 61
Figure 2.20: Summary of viral vector advantages and disadvantages 65
Figure 3.21: The application of TCA in gene expression 77
Figure 3.22: Optimization of lead compounds with siRNAs 82
Figure 3.23: Comparison of gene expression profiles to optimize lead compounds 83
Figure 3.24: Investigation of the intracellular mechanism of Endothelin A receptor 85
Figure 3.25: Schematic of knock-out and knock-down transgenics 87
Figure 3.26: Heritable suppression of Neil-1 in mouse model 88
Figure 3.27: ArteMiceTM RNAi in vivo in 4 months 89
Figure 3.28: Artemis Pharmaceutical timelines for transgenic animals 89
Figure 3.29: Status leptinR knockdown using shRNAs 91
Figure 3.30: Impact of RNAi in R&D 92
Figure 4.31: Antisense drugs currently in clinical development 98
Figure 4.32: RNAi drugs currently in clinical development 100
Figure 4.33: Development of AMD 102
Figure 4.34: siRNA targeting VEGF reduces blood vessel growth in the cornea 103
Figure 4.35: Lead siRNA candidates block HCV replication 109
Figure 4.36: HCV target destruction in mouse liver 110
Figure 4.37: Efficacy of HIV drug in vitro 112
Figure 4.38: In vivo efficacy of direct RNAi for RSV 115
Figure 4.39: Systemic siRNA leads to significant reduction in apolipoproteins 134
Figure 5.40: Conventional RISC silencing pathways and RISC pathway using “On-Target” siRNA
reagents 144
Figure 5.41: siRNA RISC process using “On-Target plus” siRNA Reagents 145
Figure 5.42: Schematic representation of aptazyme development 152
Figure 6.43: Key RNA-based companies targeting RNAi reagents 157
Figure 6.44: Key RNA-based companies targeting therapeutic agents 159
Figure 6.45: Sirna Therapeutics’ IP portfolio and therapeutic areas 167
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Figure 7.46: RNAi market segments, 2004 181
Figure 7.47: Growth in the RNAi market 2004-2010 181
Figure 7.48: Alliances in RNAi R&D 184
Figure 7.49: Potential value of therapy areas targeted by RNAi therapeutics, 2004 & 2010 186
List of Tables
Table 1.1: Advantages and disadvantages of OGN technology 26
Table 1.2: Advantages and disadvantages of modified OGNs 29
Table 1.3: Advantages and disadvantages of TFOs 31
Table 1.4: Advantages and disadvantages of ribozymes 32
Table 1.5: Advantages and disadvantages of DNAzymes 32
Table 1.6: Advantages and disadvantages of aptamers 33
Table 1.7: Genes crucial for RNAi in model organisms 35
Table 1.8: Advantages of RNAi 37
Table 1.9: Disadvantages of RNAi 38
Table 2.10: Algorithms available for designing siRNAs 44
Table 2.11: Class of functional RNA molecule 45
Table 2.12: Companies offering siRNA synthesis 48
Table 2.13: Advantages of ddRNAi versus siRNA 55
Table 2.14: Advantages of eiRNA versus siRNAs 56
Table 3.15: Commercial siRNA libraries 79
Table 4.16: Antiviral siRNA targets 108
Table 4.17: RNAi-based targeted therapies 119
Table 4.18: Examples of RNAi targets for neuronal pain 125
Table 4.19: Chemotherapeutic siRNA targets 128
Table 5.20: Animal miRNA genes with genetically assigned functions 147
Table 6.21: RNA patents registered worldwide up to March 2005 155
Table 7.22: Companies involved in RNAi technologies, A-M 179
Table 7.23: Companies involved in RNAi technologies, N-Z 180
Table 7.24: Sales forecasts for total RNAi market, 2004-2015 182
Table 7.25: Sales forecasts for siRNA synthesis and delivery, 2004-2015 183
Table 7.26: Sales forecasts for RNAi reagents, 2004-2015 183
Table 7.27: Sales forecasts for RNAi in drug discovery & target validation, 2004-2015 185
Table 7.28: Sales forecasts for RNAi therapeutics, 2004-2015 187
Table 7.29: Sales forecasts for RNAi therapeutic drugs launched 2010-2015 188
