RNAi - technologies, markets and companies |
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0. Executive Summary .... 11 1. Technologies for suppressing gene function.... 13 Introduction...... 13 RNA13 Non-coding RNA13 RNA research and potential applications....14 Role of RNA in regulation of the dihydrofolate reductase gene......14 Gene regulation 15 Post-transcriptional regulation of gene expression ...........15 Alternative RNA splicing ..........16 Technologies for gene suppression ......... 17 Antisense oligonucleotides.......17 Transcription factor decoys......18 Aptamers .........18 Ribozymes .......18 Aptazymes .......19 RNA aptamers vs allosteric ribozymes.......19 RNA Lasso........19 Peptide nucleic acid 20 PNA-DNA chimeras.20 Locked nucleic acid.21 Gene silencing ..21 Post-transcriptional gene silencing ...........21 TargeTron Ô technology for gene knockout 21Definitions and terminology of RNAi ....... 22 RNAi mechanisms ... 22 Piwi-interacting RNAs in germ cell development.......... 24 Relation of RNAi to junk DNA .. 24 RNA editing and RNAi.............. 24 Historical landmarks in the development of RNAi ........ 25 2. RNAi Technologies ...... 27 Introduction...... 27 Comparison of antisense and RNAi.......... 27 Advantages of antisense over siRNAs........27 Advantages of siRNAs over antisense........28 RNA aptamers vs siRNA ..........28 RNA Lassos versus siRNA ........28 Antisense vs DNP-ssRNA and DNP-siRNA 29 LNA and RNAi..... 30 LNA for gene suppression........30 Comparison of LNA and RNAi ...30 Use of siLNA to improve siRNA.30 RNAi versus small molecules... 30 RNAi kits ............ 31 ShortCut™ RNAi Kit 31 HiScribe™ RNAi Transcription Kit..............32 pSUPER RNAi system..............32 Si2 Silencing Duplex...............33 Techniques for measuring RNAi-induced gene silencing .............. 33 Application of PCR in RNAi.......33 Real-time quantitative PCR.34 Assessment of the silencing effect of siRNA by RT-PCR34 Bioinformatics tools for design of siRNAs 35 Random siRNA design.............35 Rational siRNA design .............35 The concept of pooling siRNAs ............36 Criteria for rational siRNA design.........37 BLOCK-iT RNAi Designer ....37 QIAGEN's 2-for-Silencing siRNA Duplexes..37 Designing vector-based siRNA.............37 iRNAChek for designing siRNA.............38 TROD: T7 RNAi Oligo Designer............38 siDirect: siRNA design software...........38 Prediction of efficacy of siRNAs 38 Algorithms for prediction of siRNA efficacy.38 - 4 - siRNA database.39 Production of siRNAs .............. 39 Chemical synthesis of short oligonucleotides...39 In vitro transcription...............40 Generation of siRNA in vivo .....40 siRNA:DNA hybrid molecules ...41 Modifications of siRNAs ...........41 Synthetic RNAs vs siRNAs ..42 Specificity of siRNAs ............... 42 Genome-wide data sets for the production of esiRNAs. 42 ddRNAi for inducing RNAi........ 43 ddRNAi technology .43 Advantages of ddRNAi over siRNA ............43 Short hairpin RNAs.. 44 Advantages and limitations of shRNA........45 Expressed interfering RNA ...... 45 RNA-induced transcriptional silencing complex ........... 46 Inhibition of gene expression by antigene RNA ........... 47 RNAi vs mRNA modulation by small molecular weight compounds ... 47 3. MicroRNA.... 49 Introduction...... 49 miRNA and RISC 50 Role of the microprocessor complex in miRNA............. 51 miRNAs compared to siRNAs... 52 Mapping miRNA genes ............ 52 Role of miRNA in gene regulation ........... 53 Control of gene expression by miRNA .......54 miRNA-mediated translational repression involving Piwi ..............54 Role of miRNAs in gene regulation during stem cell differentiation54 Mechanism of miRNAs-induced silencing of gene expression .............54 Detection and expression profiling of miRNA ............... 55 Real-time PCR for expression profiling of miRNAs............55 Use of LNA to explore miRNA...56 Microarrays for analysis of miRNA gene expression..........56 Computational approaches for the identification of miRNAs ........ 57 Biochemical approach to identification of miRNA......... 57 Targeting of miRNAs with antisense oligonucleotides.. 57 Silencing miRNAs by antagomirs............. 58 miRNA-regulated lentiviral vectors ......... 58 miRNAs as drug targets .......... 58 Prediction of miRNA targets.....59 Role of miRNA in human health and disease 60 Role of miRNAs in regulation of hematopoiesis60 Influence of miRNA on stem cell formation and maintenance ............61 Role of miRNA in regulation of aging.........61 Role of miRNA in inflammation.62 Role of miRNA in the cardiovascular system 62 miRNAs as potential therapeutic targets in heart disease..62 miRNA-based approach for reduction of hypercholesterolemia ..........62 Role of miRNA in neurological disorders . 63 Schizophrenia and miRNA .......63 Role of miRNA in viral infections............. 63 Role of miRNA in HSV-1 latency ...............63 miRNA and skin disorders ....... 64 Role of miRNA in inflammatory skin disorders .64 Role of miRNAs in cancer ........ 64 Role of miRNAs in viral oncogenesis..........65 miRNA genes in cancer ...........65 Cancer miRNA signature .........66 miRNA biomarkers in cancer....66 Diagnostic value of miRNA in cancer.........67 miRNA and brain cancer.....67 miRNA and breast cancer ...67 miRNA and colorectal cancer...............68 miRNA and hematological malignancies68 miRNA and lung cancer ......69 miRNA and pancreatic cancer .............69 miRNA and prostatic cancer70 miRNA and thyroid cancer ..70 miRNAs as basis of cancer therapeutics.....70 - 5 - miRNA gene as a basis of cancer therapy ..70 Future prospects of miRNA ..... 70 4. Methods of delivery in RNAi............. 73 Introduction...... 73 Methods of delivery of oligonucleotides .. 73 Oral and rectal administration..74 Pulmonary administration........74 Targeted delivery to the CNS...74 High flow microinfusion into the brain parenchyma.....75 Intracellular guidance by special techniques ...75 Biochemical microinjection ......76 Liposomes-mediated oligonucleotide delivery..76 Polyethylenimine-mediated oligonucleotide delivery.........76 Delivery of TF Decoys .............76 Biodegradable microparticles ...77 Microparticles ...77 Nanoparticles....77 siRNA delivery technologies.... 77 Local delivery of siRNA............78 In vivo delivery of siRNAs by synthetic vectors78 Intracellular delivery of siRNAs 79 Protein transduction domains..............79 MPG-based delivery of siRNA ..............79 Delivery of siRNAs with aptamer-siRNA chimeras .......80 Phosphorothioate stimulated cellular delivery of siRNA80 Targeted delivery of siRNAs by lipid-based technologies...80 Systemic in vivo delivery of lipophilic siRNAs .............81 NeoLipid™ technology........81 siFECTamine Ô ..81Delivery of siRNA-lipoplexes ...............81 Electroporation .82 Nucleofactor technology .....82 Intravascular delivery of siRNA 83 27mer siRNA duplexes for improved delivery and potency83 TransIT-TKO Ò...84DNA-based plasmids for delivery of siRNA .85 Convergent transcription....85 PCR cassettes expressing siRNAs .............85 Genetically engineered bacteria for delivery of shRNA......86 Viral vectors for delivery of siRNA ............86 Retroviral delivery of siRNA 86 Adenoviral vectors.............86 Lentiviral vectors...............87 Recombinant baculovirus vector..........88 Adeno-associated virus vectors for shRNA expression ......88 Delivery of siRNA without a vector............88 Cell-penetrating peptides for delivery of siRNAs ..............89 Role of nanobiotechnology in siRNA delivery...89 Quantum dots to monitor siRNA delivery ...89 Polyethylenimine nanoparticles for siRNA delivery ......90 Chitosan-coated nanoparticles for siRNA delivery .......90 siRNA delivery by lipid nanoparticles....91 Delivery of siRNA by nanosize liposomes ...91 Lipidic aminoglycoside as siRNA nanocarrier ..............92 Targeted delivery of siRNAs to specific cell populations ....92 siRNA Dynamic PolyConjugates for delivery to hepatocytes .........92 Delivery of siRNA to the CNS ..............92 Control of RNAi and siRNA levels ............ 93 siRNA pharmacokinetics in mammalian cells ............... 93 Mathematical modeling for determining the dosing schedule of siRNA 94 5. RNAi in Research ........ 95 Introduction...... 95 Basic RNAi research95 Genes and lifespan ............95 Antiviral role of RNAi in animal cells.....95 Silencing snoRNA genes .....95 Profiling small RNAs by Massively Parallel Signature Sequencing ..96 Study of signaling pathways ...............96 RNAi for research in neuroscience .......97 - 6 - Use of RNAi to study insulin action ......97 Detection of cancer mutations.............97 Loss-of-function genetic screens .........97 Inducible and reversible RNAi .............98 Combination of siRNA with green fluorescent protein ..98 RNAi and environmental research........99 Applied RNAi research............. 99 RNAi for gene expression studies .............99 Microarrays for measuring gene expression in RNAi .........99 RNAi for functional genomic analysis ...... 100 RNAi studies on C. elegans ............... 100 RNAi studies on Drosophila............... 101 RNAi in planaria .............. 102 Testing the specificity of RNAi 102 Tissue-specific RNAi.............. 102 siRNA-mediated gene silencing .............. 103 RNAi libraries.. 103 Universal plasmid siRNA library......... 104 pDual library using plasmid vector..... 104 pHippy plasmid vector library ........... 104 siRNA libary including miRNAs .......... 105 siRNA libraries using pRetroSuper vector. 105 siRNA produced by enzymatic engineering of DNA .... 105 shRNA libraries ............... 105 Enzymatic production of RNAi library . 106 RNAi and alternative splicing . 107 RNAi in animal development .. 107 RNAi for creating transgenic animals.. 107 RNAi for creating models of neurological disorders.... 108 Research support for RNAi in US........... 109 RNAi for toxicogenomics ....... 109 Role of RNAi in the US biodefense research .. 109 The RNAi Consortium............ 109 Research support for RNAi in Europe .... 110 European Union for RNA Interference Technology.......... 110 Research support of RNAi...... 110 Role of RNAi in MitoCheck project........... 111 Genome-Wide RNAi Global Initiative..... 111 6. RNAi in drug discovery .. 113 Basis of RNAi for drug discovery............. 113 Use of siRNA libraries to identify genes as therapeutic targets ........ 113 Role of siRNAs in drug target identification .............. 113 Use of a genome-wide, siRNA library for drug discovery ............ 114 Use of arrayed adenoviral siRNA libraries for drug discovery ...... 114 RNAi as a tool for assay development ..... 114 Targeting human kinases with an siRNAi library ............ 115 Challenges of drug discovery with RNAi... 115 Express TrackSM siRNA Drug Discovery Program ............ 115 Genome-wide siRNA screens in mammalian cells........... 116 Natural antisense and ncRNA as drug targets 116 MicroRNAs as drug targets .... 117 RNAi for target validation ...... 117 Delivering siRNA for target validation in vivo ........... 117 Off-target effects of siRNA-mediated gene silencing.. 119 Bioinformatic approach to off-target effects ............. 120 Selection of siRNA versus shRNA for target validation ............... 120 Application of RNAi to the druggable genome 121 Application of siRNA during preclinical drug development ............... 121 siRNAs vs small molecules as drugs........ 121 siRNAs vs antisense drugs..... 122 RNAi technology in plants for drug discovery and development ....... 123 Application of RNAi to poppy plant as source of new drugs ........ 123 7. Therapeutic applications of RNAi ... 125 Introduction.... 125 Potential of RNAi-based therapies ........ 126 In vitro applications of siRNA 126 In vivo applications of RNAi .. 127 RNAi and cell therapy............ 127 Gene inactivation to study hESCs........... 127 - 7 - RNAi and stem cells.............. 128 Cell therapy for immune disorders.......... 129 RNAi gene therapy 129 Drug-inducible systems for control of gene expression... 129 Potential side effects of RNAi gene therapy... 130 Systemic delivery of siRNAs... 130 In vivo RNAi therapeutic efficacy in animal models of human diseases .. 131 Virus infections 131 RNAi approaches to viral infections......... 132 RNAi applications in HIV........ 133 siRNA-directed inhibition of HIV-1 infection ............. 133 Role of the nef gene during HIV-1 infection and RNAi 134 Bispecific siRNA constructs ............... 134 Targeting CXCR4 with siRNAs ........... 135 Targeting CCR5 with siRNAs ............. 135 Synergistic effect of snRNA and siRNA .... 135 Anti-HIV shRNA for AIDS lymphoma .. 136 Influenza....... 136 Inhibition of influenza virus by siRNAs .... 137 Delivery of siRNA in influenza ........... 137 Challenges and future prospects of siRNAs for influenza ............ 138 Respiratory syncytial and parainfluenza viruses............. 138 Coronavirus/severe acute respiratory syndrome............ 139 Herpes simplex virus 2.......... 140 Hepatitis B ..... 140 Hepatitis C virus .. 140 Cytomegalovirus .. 142 siRNA vs antisense oligonucleotides for viral infections... 142 RNAi-based rational approach to antimalarial drug discovery.... 143 RNAi applications in oncology ............... 143 RNAi approach to study TRAIL ............... 145 Modification of alternative splicing in cancer . 145 siRNA for cancer chemoprevention ......... 145 Drug discovery for cancer...... 146 Inhibition of angiogenesis...... 146 Inhibition of oncogenes......... 147 Onconase....... 147 Allele-specific inhibition......... 147 Drug delivery issues in managing cancer |
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