RNAi - technologies, markets and companies

• Publication date: February 2012

RNA interference (RNAi) or gene silencing involves the use of double stranded RNA (dsRNA). Once inside the cell, this material is processed into short 21-23 nucleotide RNAs termed siRNAs that are used in a sequence-specific manner to recognize and destroy complementary RNA. The report compares RNAi with other antisense approaches using oligonucleotides, aptamers, ribozymes, peptide nucleic acid and locked nucleic acid. Various RNAi technol...

RNA interference (RNAi) or gene silencing involves the use of double stranded RNA (dsRNA). Once inside the cell, this material is processed into short 21-23 nucleotide RNAs termed siRNAs that are used in a sequence-specific manner to recognize and destroy complementary RNA. The report compares RNAi with other antisense approaches using oligonucleotides, aptamers, ribozymes, peptide nucleic acid and locked nucleic acid.

Various RNAi technologies are described, along with design and methods of manufacture of siRNA reagents. These include chemical synthesis by in vitro transcription and use of plasmid or viral vectors. Other approaches to RNAi include DNA-directed RNAi (ddRNAi) that is used to produce dsRNA inside the cell, which is cleaved into siRNA by the action of Dicer, a specific type of RNAse III. MicroRNAs are derived by processing of short hairpins that can inhibit the mRNAs. Expressed interfering RNA (eiRNA) is used to express dsRNA intracellularly from DNA plasmids.

Delivery of therapeutics to the target tissues is an important consideration. siRNAs can be delivered to cells in culture by electroporation or by transfection using plasmid or viral vectors. In vivo delivery of siRNAs can be carried out by injection into tissues or blood vessels or use of synthetic and viral vectors.

Because of its ability to silence any gene once the sequence is known, RNAi has been adopted as the research tool to discriminate gene function. After the genome of an organism is sequenced, RNAi can be designed to target every gene in the genome and target for specific phenotypes. Several methods of gene expression analysis are available and there is still need for sensitive methods of detection of gene expression as a baseline and measurement after gene silencing. RNAi microarray has been devised and can be tailored to meet the needs for high throughput screens for identifying appropriate RNAi probes. RNAi is an important method for analyzing gene function and identifying new drug targets that uses double-stranded RNA to knock down or silence specific genes. With the advent of vector-mediated siRNA delivery methods it is now possible to make transgenic animals that can silence gene expression stably. These technologies point to the usefulness of RNAi for drug discovery.

RNAi can be rationally designed to block the expression of any target gene, including genes for which traditional small molecule inhibitors cannot be found. Areas of therapeutic applications include virus infections, cancer, genetic disorders and neurological diseases. Research at academic centers that is relevant to RNAi-based therapeutics is mentioned.

Regulatory, safety and patent issues are discussed. Side effects can result from unintended interaction between an siRNA compound and an unrelated host gene. If RNAi compounds are designed poorly, there is an increased chance for non-specific interaction with host genes that may cause adverse effects in the host. However, there are no major safety concerns and regulations are in preliminary stages as the clinical trials are still ongoing and there are no marketed products. Many of the patents are still pending.

The markets for RNAi are difficult to define as no RNAi-based product is approved yet but several are in clinical trials. The major use of RNAi reagents is in research but it partially overlaps that of drug discovery and therapeutic development. Various markets relevant to RNAi are analyzed from 2011 to 2021. Markets are also analyzed according to breakdown of technologies and use of siRNAs, miRNAs, etc.

Profiles of 161 companies involved in developing RNAi technologies are presented along with 218 collaborations. They are a mix of companies that supply reagents and technologies (nearly half of all) and companies that use the technologies for drug discovery. Out of these, 33 are developing RNAi-based therapeutics and 30 are involved in microRNAs. The bibliography contains selected 600 publications that are cited in the report. The text is supplemented with 35 tables and 10 figures.

Biological Therapy Industry

TABLE OF CONTENTS

0. Executive Summary 15
1. Technologies for suppressing gene function 17
Introduction 17
DNA transcription 17
RNA 17
Non-coding RNA 17
RNA research and potential applications 18
Role of RNA in regulation of the dihydrofolate reductase gene 19
Gene regulation 19
Post-transcriptional regulation of gene expression 20
Alternative RNA splicing 21
Technologies for gene suppression 21
Antisense oligonucleotides 21
Transcription factor decoys 22
Aptamers 22
Ribozymes 23
Aptazymes 23
RNA aptamers vs allosteric ribozymes 24
RNA Lasso 24
Peptide nucleic acid 24
PNA-DNA chimeras 25
Locked nucleic acid 25
Gene silencing 25
Post-transcriptional gene silencing 26
TargeTron? technology for gene knockout 26
Definitions and terminology of RNAi 26
RNAi mechanisms 27
Non-promoter-associated small RNAs 29
Piwi-interacting RNAs in germ cell development 30
Relation of RNAi to junk DNA 30
RNA editing and RNAi 31
Historical landmarks in the development of RNAi 31
2. RNAi Technologies 33
Introduction 33
Comparison of antisense and RNAi 33
Advantages of antisense over siRNAs 33
Advantages of siRNAs over antisense 34
RNA aptamers vs siRNA 34
RNA Lassos versus siRNA 34
Concluding remarks on antisense vs RNAi 35
ssRNAi 35
Antisense vs DNP-ssRNA and DNP-siRNA 35
LNA and RNAi 36
LNA for gene suppression 36
Comparison of LNA and RNAi 37
Use of siLNA to improve siRNA 37
RNAi versus small molecules 37
RNAi in vivo 37
Cre-regulated RNAi in vivo 38
RNAi kits 38
ShortCut™ RNAi Kit 38
HiScribe™ RNAi Transcription Kit 39
pSUPER RNAi system 39
Si2 Silencing Duplex 40
Techniques for measuring RNAi-induced gene silencing 40
Application of PCR in RNAi 40
Real-time quantitative PCR 41
Assessment of the silencing effect of siRNA by RT-PCR 41
Fluorescence resonance energy transfer probe for RNA interactions 42
Bioinformatics tools for design of siRNAs 42
Random siRNA design 42
Rational siRNA design 43
The concept of pooling siRNAs 44
Criteria for rational siRNA design 44
BLOCK-iT RNAi Designer 44
QIAGEN's 2-for-Silencing siRNA Duplexes 45
Designing vector-based siRNA 45
iRNAChek for designing siRNA 45
TROD: T7 RNAi Oligo Designer 45
siDirect: siRNA design software 46
Prediction of efficacy of siRNAs 46
Algorithms for prediction of siRNA efficacy 46
siRNA databases 46
Production of siRNAs 47
Chemical synthesis of short oligonucleotides 47
In vitro transcription 47
Generation of siRNAs in vivo 48
UsiRNAs 48
siRNA:DNA hybrid molecules 49
Chemical modifications of siRNAs 49
Sugar modifications of siRNA 49
Phosphate linkage modifications of siRNA 49
Modifications to the siRNA overhangs 50
Modifications to the duplex architecture 50
Applications of chemical modification of siRNAs 50
Synthetic RNAs vs siRNAs 51
Specificity of siRNAs 51
Asymmetric interfering RNA 52
Genome-wide data sets for the production of esiRNAs 52
ddRNAi for inducing RNAi 52
ddRNAi technology 52
Advantages of ddRNAi over siRNA 53
Short hairpin RNAs 54
siRNA versus shRNA 54
Circular interfering RNA 55
Expressed interfering RNA 56
RNA-induced transcriptional silencing complex 56
Inhibition of gene expression by antigene RNA 57
RNAi vs mRNA modulation by small molecular weight compounds 57
3. MicroRNA 59
Introduction 59
miRNA and RISC 61
Role of the microprocessor complex in miRNA 61
miRNAs compared to siRNAs 62
miRNA and stem cells 63
Influence of miRNA on stem cell formation and maintenance 63
Role of miRNAs in gene regulation during stem cell differentiation 63
miRNA databases 64
Sanger miRBase miRNA sequence database 64
Mapping miRNA genes 64
A database of ultraconserved sequences and miRNA function 65
A database for miRNA deregulation in human disease 65
An database of miRNA-target interactions 66
Role of miRNA in gene regulation 66
Control of gene expression by miRNA 67
miRNA-mediated translational repression involving Piwi 67
Transcriptional regulators of ESCs control of miRNA gene expression 67
Mechanism of miRNAs-induced silencing of gene expression 67
miRNA diagnostics 68
Biochemical approach to identification of miRNA 68
Computational approaches for the identification of miRNAs 69
LNA probes for exploring miRNA 69
Microarrays for analysis of miRNA gene expression 69
Microarrays vs quantitative PCR for measuring miRNAs 70
miRNAs as biomarkers of hepatotoxicity 70
Modification of in situ hybridization for detection of miRNAs 71
Nuclease Protection Assay to measure miRNA expression 71
Real-time PCR for expression profiling of miRNAs 71
Targeting of miRNAs with antisense oligonucleotides 72
Silencing miRNAs by antagomirs 72
New tools for miRNA silencing 72
Use of HAPIscreen for identification of aptamers against pre-miRNAs 73
miRNA-regulated lentiviral vectors 73
miRNAs as drug targets 73
miRNAs as targets for antisense drugs 74
Challenges facing use of miRNAs as drug targets 74
Target specificity of miRNAs 75
Prediction of miRNA targets 75
Role of miRNA in human health and disease 76
Role of miRNAs in regulation of hematopoiesis 76
Role of miRNA depletion in tissue regeneration 76
Role of miRNA in regulation of aging 77
Role of miRNA in inflammation 77
Role of miRNAs in regulation of immune system 77
Role of miRNA in the cardiovascular system 78
Role of miRNAs in development of the cardiovascular system 78
Role of miRNAs in angiogenesis 78
Role of miRNAs in cardiac hypertrophy and failure 78
Role of miRNAs in conduction and rhythm disorders of the heart 79
Diagnostic and prognostic value of miRNAs in acute coronary syndrome 79
miRNA-based approaches for reduction of hypercholesterolemia 80
miRNA-based approach for restenosis following angioplasty 80
miRNA gene therapy for ischemic heart disease 80
miRNAs as therapeutic targets for cardiovascular diseases 81
Concluding remarks and future prospects of miRNA in the cardiovascular system 81
Role of miRNAs in the nervous system 81
miRNAs and addiction 82
miRNAs in neurodegenerative disorders 82
miRNAs as biomarkers of Alzheimer’s disease 83
miRNAs in Huntington’s disease 83
miRNA malfunction in spinal motor neuron disease 83
miRNAs and retinal neurodegenerative disorders 84
miRNA and schizophrenia 84
Role of miRNA in viral infections 84
Role of miRNA in HSV-1 latency 84
miRNA and autoimmune disorders 85
miRNA in rheumatoid arthritis 85
miRNA in systemic lupus erythematosus 85
miRNAs in gastrointestinal disorders 86
miRNA-based therapies for the irritable bowel syndrome 86
miRNA and skin disorders 86
Role of miRNA in inflammatory skin disorders 86
Role of miRNAs in cancer 86
miRNAs linked to the initiation and progression of cancer 86
Oncomirs 87
Linking miRNA sequences to cancer using RNA samples 88
Role of miRNAs in viral oncogenesis 88
miRNA genes in cancer 88
miRNAs interaction with p53 89
miRNAs, embryonic stem cells and cancer 89
miRNAs and cancer metastases 90
Role of miRNAs in cancer diagnosis 91
Cancer miRNA signature 91
miRNA biomarkers in cancer 91
Diagnostic value of miRNA in cancer 92
Prognostic value of miRNA in cancer 92
miRNAs as basis of cancer therapeutics 92
Antisense oligonucleotides targeted to miRNA 92
Delivery of miRNA mimetics in Cancer 93
Role of miRNAs in adoptive immunotherapy of cancer 93
Restoration of tumor suppressor miRNA may inhibit cancer 93
Role of miRNAs in various cancers 94
miRNA and brain cancer 94
miRNA and breast cancer 95
miRNA and colorectal cancer 95
miRNA and gastrointestinal cancer 95
miRNA and hematological malignancies 96
miRNA and hepatocellular carcinoma 97
miRNA and lung cancer 97
miRNA and nasopharyngeal carcinoma 98
miRNA and ovarian cancer 99
miRNA and pancreatic cancer 99
miRNA and prostatic cancer 100
miRNA and thyroid cancer 100
Future prospects of miRNA therapeutics 101
Companies involved in miRNA 101
4. Methods of delivery in RNAi 105
Introduction 105
Methods of delivery of oligonucleotides 105
Oral and rectal administration 106
Pulmonary administration 106
Targeted delivery to the CNS 106
High flow microinfusion into the brain parenchyma 107
Intracellular guidance by special techniques 107
Biochemical microinjection 108
Liposomes-mediated oligonucleotide delivery 108
Polyethylenimine-mediated oligonucleotide delivery 108
Delivery of TF Decoys 108
Biodegradable microparticles 109
Microparticles 109
Nanoparticles 109
Self-delivering rxRNA 109
siRNA delivery technologies 110
Local delivery of siRNA 111
In vivo delivery of siRNAs by synthetic vectors 111
Intracellular delivery of siRNAs 111
Delivery of siRNAs with aptamer-siRNA chimeras 112
MPG-based delivery of siRNA 112
Nanoparticles for intracellular delivery of siRNA 112
Protamine-antibody fusion proteins for delivery of siRNA to cells 112
Protein transduction domains 113
Phosphorothioate stimulated cellular delivery of siRNA 113
Targeted delivery of siRNAs by lipid-based technologies 113
Delivery of siRNA-lipoplexes 114
Lipidoids for delivery of siRNAs 114
NeoLipid™ technology 115
siFECTamine? 115
Systemic in vivo delivery of lipophilic siRNAs 115
Systemic delivery of siRNAi by lipid nanoparticles 115
Challenges and future prospects of lipid-based siRNA delivery 116
Electroporation 116
Nucleofactor technology 117
Visualization of electrotransfer of siRNA at single cell level 117
Intravascular delivery of siRNA 117
27mer siRNA duplexes for improved delivery and potency 118
TransIT-TKO? 118
DNA-based plasmids for delivery of siRNA 119
Convergent transcription 120
PCR cassettes expressing siRNAs 120
Genetically engineered bacteria for delivery of shRNA 120
Viral vectors for delivery of siRNA 120
Adenoviral vectors 120
Adeno-associated virus vectors for shRNA expression 121
Baculovirus vector 121
Lentiviral vectors 122
Retroviral delivery of siRNA 123
Transkingdom RNAi delivery by genetically engineered bacteria 123
Delivery of siRNA without a vector 123
Cell-penetrating peptides for delivery of siRNAs 124
Role of nanobiotechnology in siRNA delivery 124
Chitosan-coated nanoparticles for siRNA delivery 124
Cyclodextrin nanoparticles 125
Delivery of gold nanorod-siRNA nanoplex to dopaminergic neurons 125
Lipidic aminoglycoside as siRNA nanocarrier 125
Lipid nanoparticles-mediated siRNA delivery 125
Nanosize liposomes for delivery of siRNA 126
PAMAM dendrimers for siRNA delivery 126
Polyethylenimine nanoparticles for siRNA delivery 127
Polycation-based nanoparticles for siRNA delivery 127
Quantum dots to monitor siRNA delivery 128
Targeted delivery of siRNAs to specific organs 128
siRNA delivery to the CNS 128
siRNA delivery to the liver 129
siRNA delivery to the lungs 129
Control of RNAi and siRNA levels 130
siRNA pharmacokinetics in mammalian cells 130
Mathematical modeling for determining the dosing schedule of siRNA 131
Assessing siRNA pharmacodynamics in animal models 131
Research on siRNA delivery funded by the NIH 131
Companies involved in delivery technologies for siRNA 132
5. RNAi in Research 135
Introduction 135
Basic RNAi research 135
Antiviral role of RNAi in animal cells 135
Combination of siRNA with green fluorescent protein 135
Detection of cancer mutations 136
Genes and lifespan 136
Inducible and reversible RNAi 136
Loss-of-function genetic screens 136
Profiling small RNAs 137
RNAi for research in neuroscience 137
RNAi and environmental research 138
Silencing snoRNA genes 138
Study of signaling pathways 138
Transgenic RNAi 139
Use of RNAi to study insulin action 139
Applied RNAi research 139
RNAi for gene expression studies 139
Microarrays for measuring gene expression in RNAi 139
RNAi for functional genomic analysis 140
RNAi studies on C. elegans 140
RNAi studies on Drosophila 141
RNAi in planaria 142
Testing the specificity of RNAi 142
Tissue-specific RNAi 142
siRNA-mediated gene silencing 143
RNAi libraries 143
Universal plasmid siRNA library 144
pDual library using plasmid vector 144
pHippy plasmid vector library 145
siRNA libary including miRNAs 145
siRNA libraries using pRetroSuper vector 145
siRNA produced by enzymatic engineering of DNA 145
shRNA libraries 146
Enzymatic production of RNAi library 147
RNAi and alternative splicing 147
RNAi in animal development 147
RNAi for creating transgenic animals 148
RNAi for creating models of neurological disorders 148
Research support for RNAi in US 149
RNAi for toxicogenomics 149
Role of RNAi in the US biodefense research 149
The RNAi Consortium 149
Research support for RNAi in Europe 150
European Union for RNA Interference Technology 150
Research support of RNAi 151
Role of RNAi in MitoCheck project 151
RNAi Global Initiative 152
SIROCCO project 153
6. RNAi in drug discovery 155
Basis of RNAi for drug discovery 155
RNAi for identification of genes as therapeutic targets 155
Role of siRNAs in drug target identification 156
Use of a genome-wide, siRNA library for drug discovery 156
Use of arrayed adenoviral siRNA libraries for drug discovery 156
RNAi as a tool for assay development 157
Targeting human kinases with an siRNAi library 157
Challenges of drug discovery with RNAi 157
Express TrackSM siRNA Drug Discovery Program 158
Genome-wide siRNA screens in mammalian cells 158
PhenomicID™ 158
Natural antisense and ncRNA as drug targets 159
RNAi for target validation 159
Delivering siRNA for target validation in vivo 159
Off-target effects of siRNA-mediated gene silencing 161
Bioinformatic approach to off-target effects 162
Validation of oncology targets discovered through RNAi screens 162
Selection of siRNA versus shRNA for target validation 163
Application of RNAi to the druggable genome 163
Application of siRNA during preclinical drug development 163
siRNAs vs small molecules as drugs 164
siRNAs vs antisense drugs 164
RNAi technology in plants for drug discovery and development 165
Application of RNAi to poppy plant as source of new drugs 165
7. Therapeutic applications of RNAi 167
Introduction 167
Potential of RNAi-based therapies 168
In vitro applications of siRNA 168
In vivo applications of RNAi 169
RNAi and cell therapy 169
Gene inactivation to study hESCs 170
RNAi and stem cells 170
Cell therapy for immune disorders 171
RNAi gene therapy 171
Drug-inducible systems for control of gene expression 171
Potential side effects of RNAi gene therapy 172
Systemic delivery of siRNAs 172
In vivo RNAi therapeutic efficacy in animal models of human diseases 173
Virus infections 173
RNAi approaches to viral infections 174
Delivery of siRNAs in viral infections 175
RNAi applications in HIV 175
A multiple shRNA approach for silencing of HIV-1 176
Anti-HIV shRNA for AIDS lymphoma 176
Aptamer-mediated delivery of anti-HIV siRNAs 176
Bispecific siRNA constructs 176
Role of the nef gene during HIV-1 infection and RNAi 177
siRNA-directed inhibition of HIV-1 infection 177
Synergistic effect of snRNA and siRNA 178
Targeting CXCR4 with siRNAs 178
Targeting CCR5 with siRNAs 178
Concluding remarks on RNAi approach to HIV/AIDS 179
Influenza 179
Inhibition of influenza virus by siRNAs 180
Delivery of siRNA in influenza 181
Challenges and future prospects of siRNAs for influenza 181
Respiratory syncytial and parainfluenza viruses 182
Coronavirus/severe acute respiratory syndrome 183
Herpes simplex virus 2 183
Hepatitis B 183
Hepatitis C virus 184
Cytomegalovirus 186
Ebola virus 186
siRNA vs antisense oligonucleotides for viral infections 186
siRNA against methicillin-resistant S. aureus 187
RNAi-based rational approach to antimalarial drug discovery 187
Inhibiting the growth of malarial parasite by heme-binding DNA aptamers 187
siRNA-based antimalarial therapeutics 188
RNAi applications in oncology 188
Allele-specific inhibition 189
Drug delivery issues in managing cancer by RNAi approach 189
Inhibition of oncogenes 190
Modification of alternative splicing in cancer 191
Onconase 191
Overcoming drug resistance in cancer 192
Targeting fusion proteins in cancer 193
Increasing chemosensitivity by RNAi 193
RNAi approach to study TRAIL 193
RNAi-based logic circuit for identification of specific cancer cells 194
siRNAs for anticancer drug discovery 194
siRNAs for inducing cancer immunity 195
siRNAs for inhibition of angiogenesis 195
siRNA targeting the R2 subunit of ribonucleotide reductase 196
siRNA for cancer chemoprevention 196
siHybrids vs siRNAs as anticancer agents 196
Nanobiotechnology-based delivery of siRNAs 197
Lipid nanoparticle-based delivery of anticancer siRNAs 197
Minicells for targeted delivery of nanoscale anticancer therapeutics 197
Nanoimmunoliposome-based system for targeted delivery of siRNA 198
Polymer nanoparticles for targeted delivery of anticancer siRNA 198
RNA nanotechnology for delivery of cancer therapeutics 199
Targeted delivery of a nanoparticle-siRNA complex in cancer patients 199
RNAi-based treatment of various cancer types 200
RNAi-based therapy of brain cancer 200
RNAi in breast cancer 201
RNAi for enhancing hyperthermia/chemotherapy in cervical cancer 202
RNAi and colorectal cancer 202
RNAi and Ewing’s sarcoma 203
RNAi and leukemias 203
RNAi and lung cancer 204
RNAi and melanoma 204
RNAi and pancreatic cancer 205
RNAi and prostate cancer 205
Genetic disorders 205
RNAi for skin disorders 206
Experimental studies for RNAi applications in skin disorders 206
Clinical applications of RNAi in skin disorders 207
Pachyonychia congenita 207
Neurological disorders 207
RNAi for neurodegenerative disorders 208
Alzheimer's disease 209
Parkinson's disease 209
Amyotrophic lateral sclerosis 210
Prion diseases 211
Polyglutamine-induced neurodegeneration 211
Fragile X syndrome and RNAi 212
RNAi-based therapy for Huntington's disease 212
Combination of RNAi and gene therapy to prevent neurodegenerative disease 213
Role of RNAi in pain therapy 214
Role of RNAi in repair of spinal cord injury 214
Role of RNAi in treatment of multiple sclerosis 215
siRNA for Duchenne muscular dystrophy 215
siRNA for dystonia 215
RNAi in ophthalmology 216
Age related macular degeneration 216
Current treatment of AMD 216
RNAi-based treatments for AMD 217
Diabetic retinopathy 218
Retinitis pigmentosa 219
RNAi and metabolic disorders 219
RNAi and obesity 220
Genes and regulation of body fat 220
RNAi and diabetes 220
Regulation of insulin secretion by a miRNA 220
RNAi for study of genes in animal models of diabetes 221
RNAi for drug discovery in diabetes 221
RNAi for treating liver dysfunction in diabetes 222
siRNAs for study of glucose transporter 222
siRNAs for targeting adipose inflammation in diabetes and obesity 223
RNAi in hematology 223
Stem cell-based gene therapy and RNAi for sickle cell disease 223
RNAi and disorders of the immune system 224
siRNA applications in immunology 224
Use of RNAi in transplantation 225
RNAi for cardiovascular disorders 225
RNAi for hypercholesterolemia 226
siRNA targeting NADPH oxidase in cardiovascular diseases 226
siRNA for study and treatment of ischemia-reperfusion injury 227
RNAi in respiratory disorders 227
siRNA for cystic fibrosis 227
siRNA for asthma 228
RNAi for musculoskeletal disorders 228
RNAi for rheumatoid arthritis 228
RNAi for bone disorders 229
RNAi for treatment of osteoporosis 229
Research relevant to RNAi-based therapies at academic institutes 230
Laboratory of RNA Molecular Biology, The Rockefeller University 230
RNAi Center, La Jolla Institute for Allergy & Immunology 230
Clinical trials of RNAi-based therapies 231
Improving efficacy of siRNAs for clinical trials by improved delivery 232
Role of RNAi in development of personalized medicine 232
Future prospects of RNAi 233
Challenges for the development of RNAi-based therapeutics 233
8. Safety, regulatory and patent issues 235
Introduction 235
Limitations and drawbacks of RNAi 235
Adverse effects of RNAi 235
Effect of siRNAs on interferon response 236
Detection of interferon response 236
Prevention of the interferon response in RNAi 237
Overcoming the innate immune response to siRNAs 237
Toxicity associated with RNAi 238
Selection of siRNAs to improve specificity and efficacy 238
Regulatory issues relevant to RNAi 238
RNAi patents 239
Companies with strong patent position 239
Alnylam 239
Benitec 242
Intradigm 242
Quark Pharmaceuticals 242
Sirna Therapeutics 243
9. Markets for RNAi Technologies 245
Introduction 245
Current and future market potential for RNAi technologies 245
RNAi reagents 246
siRNA markets 246
RNAi-based drug discovery and target validation 246
RNAi-based development of therapeutics 246
RNAi market potential according to therapeutic areas 247
Market for viral infections 247
Market for cancer 248
Market for age related macular degeneration 248
Unmet needs in RNAi 248
Strategies for marketing RNAi 249
Choosing optimal indications 249
Strategies according to the trends in healthcare in the next decade 250
Concluding remarks 251
10. Companies involved in RNAi Technologies 253
Introduction 253
Major players in RNAi 256
Profiles of companies 257
Collaborations 445
11. References 453

List of Tables
Table 1 1: Classification of small RNA molecules 27
Table 1 2: Mechanisms of small RNAs involved in gene silencing 28
Table 1 3: Historical landmarks in the evolution of RNAi 31
Table 2 1: RNAi versus small molecules 37
Table 2 2: Providers of software for siRNA design 43
Table 2 3: Methods for the production of siRNAs 47
Table 2 4: Advantages and limitations of methods of shRNA-derived siRNA knockdown 55
Table 2 5: Comparison of eiRNA with siRNA 56
Table 3 1: Methods for miRNA target prediction 75
Table 3 2: miRNA expression in neurodegenerative diseases 82
Table 3 3: Dysregulation of miRNA expression in epithelial cancers 87
Table 3 4: Companies involved in miRNA diagnostics and therapeutics 101
Table 4 1: Methods of delivery of oligonucleotides 105
Table 4 2: Methods of delivery of siRNA 110
Table 4 3: Companies developing siRNA delivery technologies 133
Table 5 1: RNAi libraries 143
Table 6 1: Delivery of siRNAs in vivo for target validation 160
Table 6 2: Selection of siRNA versus shRNA for target validation 163
Table 7 1: RNAi-based therapeutic approaches 168
Table 7 2: In vivo RNAi therapeutic efficacy in animal models of human diseases 173
Table 7 3: Inhibition of viral replication by RNAi 174
Table 7 4: Cancer-associated genes that can be targeted by RNAi 190
Table 7 5: Neurological disorders that have been studied by using RNAi 208
Table 7 6: Clinical trials of RNAi-based therapeutics 231
Table 9 1: RNAi markets according to technologies and reagents 2011-2021 245
Table 9 2: Markets for RNAi therapy for selected diseases: years 2011-2021 247
Table 10 1: RNAi reagent, technology and service companies 253
Table 10 2: Pharmaceutical companies using RNAi for drug discovery and development 254
Table 10 3: Biotechnology companies using RNAi for drug discovery and development 255
Table 10 4: Companies developing RNAi-based therapeutic products 256
Table 10 5: Major players in RNAi 256
Table 10 6: RNAi products of Benitec 277
Table 10 7: Proprietary reagents of ImuThes 332
Table 10 8: Product pipeline of Silence Therapeutics 414
Table 10 9: Collaborations in RNAi technologies 445

List of Figures
Figure 1 1: Relationship of DNA, RNA and protein in the cell 20
Figure 1 2: Schematic of suppression of gene expression by RNAi 28
Figure 2 1: Overview of ShortCut RNAi Kit 39
Figure 2 2: Gene silencing by RNAi induced with ddRNAi 53
Figure 3 1: A schematic miRNA pathway 59
Figure 3 2: Molecular mechanisms of miRNA generation 60
Figure 7 1: Targeting disease by RNAi 167
Figure 7 2: Role of RNAi in personalized medicine 233
Figure 8 1: Problems with use of synthetic siRNAs and measures to prevent them 236
Figure 9 1: Unmet needs in RNAi technologies 249