Outlook for RNAi, 2007: siRNA and miRNA in biology, diagnostics and therapeutics

Table of Contents

• Introduction
• Design and Production of RNAi effectors
• RNAi Research and Applications
• Delivering RNAi therapeutics
• RNAi Therapeutics: Progress and the Future
• miRNA: diagnostics and therapeutics
• Regulatory issues and patents
• RNAi markets and trends

• Summary
• Introduction
• DNA to RNA to protein ? a primer
• Antisense technologies
• RNA interference
• Overview and significance
• RNAi' s mechanism
  • Post-transcriptional gene silencing (PGTS) by siRNA
  • miRNA pathways
  • Transcriptional gene silencing (TGS) by siRNA
  • Other non-coding RNAs
• Advantages and disadvantages of RNAi
• Report Outline

• Summary
• Introduction
• Design of RNAi effectors
• siRNA design algorithms
  • Building silencing efficiency
  • Avoiding off-target effects
• Chemical modification
  • Avoiding immunostimulation
• Finding the right target sites
• miRNA mimics
• Production of RNAi effectors
• Chemical Synthesis
• siRNA pools
• Dicer-substrate siRNAs (DsiRNAs)
• Expression vectors
  • Plasmid expression vectors
  • Expression from a PCR product
  • Viral expression vectors
• Conclusions

• Summary
• Introduction
• Elucidating gene function
• High throughput loss-of-function screening with RNAi
• Choice of species and cell-line
• RNAi reagents
• Delivery method
  • Screening paradigm and format
• Read-out methods
• Building pathways
• Investigating gene function in vivo
• Applications of RNAi in drug discovery and development
  • Target discovery and validation
  • Optimization of drug therapy and development of personalized medicine
• miRNA Research
• miRNA Databases and Algorithms
• Identifying miRNAs, their targets and function
  • Isolation and enrichment of miRNA
  • Detection and quantification of miRNA
  • Functional analysis
• Conclusions

• Summary
• Introduction
• Direct organ delivery
• Stabilization of siRNAs
• Non-viral delivery methods
• Cholesterol and peptide conjugation
• Liposomal delivery
• Conjugation with cell penetrating peptides
• Atelocollagen
• Targeted polymeric delivery
• Aptamer-siRNA complexes
• FAb-siRNA complexes
• MAb-siRNA complexes
• Targeted nanoparticle delivery methods
  • Targeting with transferrin
  • Targeting with apatmers
  • Targeting with RGD peptide
• Delivering RNAi via Plasmid DNA
• Virosomes
• Viral delivery methods
• Conclusions

• Summary
• Introduction
• siRNA Therapeutics: Analysis by Therapeutic Area
• Ocular Diseases
• Age-related Macular Degeneration (AMD)
  • Key RNAi players
• Diabetic Retinopathy
• Viral diseases
• Respiratory Syncytial Virus (RSV)
  • Key RNAi Players
• HIV
  • Key RNAi players
• Hepatitis C Virus (HCV)
  • Key RNAi players
• Hepatitis B Virus (HBV)
  • Key RNAi players
• Pandemic Flu
  • Key RNAi players
• SARS
  • Key RNAi players
• Herpes Simplex Virus (HSV)
• Other viral diseases
• Respiratory diseases
• Asthma and Chronic Obstructive Pulmonary Disease (COPD)
  • Key RNAi Players
• Cystic fibrosis (CF)
  • Key RNAi players
• Neurological diseases
• Oncology
• Angiogenesis
  • Key RNAi players
• Oncogenes
  • Key RNAi players
• Cardiovascular disease
  • Key RNAi players
• Metabolic disorders
• Diabetes and Obesity
  • Key RNAi players
• Dermatology
  • Hair Removal
  • Key RNAi players
  • Pachyonychia congenita and related disorders
  • Key RNAi Players
• Acute renal failure
  • Key RNAi players
• Acute hearing loss/ pressure sores ototoxicity
  • Key RNAi players
• Inflammatory Diseases
  • Key RNAi players
• Conclusions

• Summary
• Introduction
• miRNA as a diagnostic tool
• Companies developing miRNA-based diagnostics
  • Rosetta Genomics
  • Cepheid
  • Stratagene
• miRNA-based therapeutics
• Companies developing miRNA-based therapeutics
  • Alnylam Pharmaceuticals
  • Asuragen Inc
  • Santaris
  • Sirna
• Conclusions

• Summary
• Introduction
• Patents in RNAi
• Seminal patents in RNAi
  • Alnylam
  • Sirna
• Chemical modifications
• DNA directed RNAi
• Patents for specific RNAi targets
• The future ? more patent litigation?
• MicroRNA
• Tuschl III patents
• Zamore and other patents
• Regulatory considerations for RNAi therapies
• Conclusions

• Summary
• Introduction
• The RNAi market
• Market size and future trends
• RNAi suppliers: synthesis and reagents
• Alliances with big pharma for RNAi in R&D
• Delivery of RNAi therapeutics
• RNAi-based therapeutics
• Big pharma alliances for RNAi-based therapeutics
• miRNA-based diagnostics
• Conclusion

• Figure 1.1: Summary of antisense technologies other than RNAi
• Figure 1.2: Timeline of RNAi discoveries
• Figure 1.3: Significance of RNAi
• Figure 1.4: Mechanism of post-transcriptional gene silencing by siRNA
• Figure 1.5: Mechanism of miRNA mediated translational repression
• Figure 2.1: Types of RNAi effector
• Figure 2.2: Points of the RNAi pathway involved in determining silencing efficiency
• Figure 2.3: Influence of guide RNA structure on siRNA efficiency
• Figure 2.4: siRNA design that avoids off-target effects
• Figure 2.5: Mechansims of immunostimulation with siRNA
• Figure 2.6: Advantages and disadvantages of siRNA synthesis methods
• Figure 2.7: Mechanism of ddRNAi compared to RNAi triggered by an siRNA
• Figure 2.8: Nucleonics multi-targeting Anti-HBV drug
• Figure 3.1: Considerations for a HT RNAi screen
• Figure 3.2: Structures of silencing reagents for RNAi screening
• Figure 3.3: Read-out methods for high throughput screens
• Figure 3.4: Method of ArteMiceTM RNAi development
• Figure 3.5: Peer reviewed miRNA publications (2001-2006)
• Figure 3.6: Comparison of Mirus' LabelIT and enzymatically labelled samples
• Figure 3.7: Inser Comparison of effectiveness of different knockdown methods t figuretitlehere,
• with date where appropriate
• Figure 4.1: Chemical modifications of siRNAs increase stability and PK
• Figure 4.2: Ribo-T siRNAs from Nastech Pharmaceuticals
• Figure 4.3: Structure of a SNALP
• Figure 4.4: Mirus Bio' s Dynamic PolyConjugates? delivery system for siRNA
• Figure 4.5: Aptamer complexes for cell-type specific delivery of siRNAs
• Figure 4.6: Heavy-chain antibody fragment for delivery of siRNAs to cell surface receptors
• Figure 4.7: Targeted nanoparticles delivering siRNA
• Figure 4.8: Aptamer directed nanoparticle formulation of docetaxel
• Figure 4.9: Preparation of virosomes encapsulating siRNA
• Figure 4.10: Viral delivery of shRNA
• Figure 5.1: Development of AMD
• Figure 5.2: siRNA targeting VEGF reduces blood vessel growth in the cornea
• Figure 5.3: Visual Acuity data from Phase 1 studies of Sirna-027
• Figure 5.4: Possible targets for suppressing HIV replication
• Figure 5.5: siRNA mediated knockdown in Sirna' s primate model of HCV chimeric infection
• Figure 5.6: Inhibition of three regions of the HCV genome by TT-033 for more than 2 months
• after a single administration to mice
• Figure 5.7: Down regulation of Hepatitis B Surface Antigen after intravenous delivery of
• Nucleonic' s HBV candidate
• Figure 6.1: Rosetta Genomics' method for miRNA identification and validation
• Figure 6.2: Illustration of Exiqon' s miRNA diagnostics
• Figure 6.3: Structure of an antagomir
• Figure 6.4: Asuragen' s development of miRNA-based therapeutics
• Figure 6.5: let-7: tumor suppressor and future therapeutic
• Figure 8.1: RNAi market sectors
• Figure 8.2: Growth in the RNAi market 2006-2015

• Table 1.1: Failures of antisense drugs
• Table 1.2: Highly generalized comparison of siRNAs and miRNAs
• Table 1.3: Advantages of RNAi
• Table 1.4: Disadvantages of RNAi
• Table 2.1: Selection of publicly available siRNA design tools
• Table 2.2: Commercial siRNA design services
• Table 2.3: Suppliers of custom and predesigned siRNAs
• Table 2.4: Suppliers of siRNA pools and kits to create them
• Table 2.5: Comparison of plasmid-based vectors and synthetic siRNA for research purposes
• Table 2.6: Selection of plasmid vector products for siRNA generation
• Table 2.7: Suppliers of kits for generating siRNA expression cassettes by PCR
• Table 2.8: Advantages and disadvantages of different viral and non-viral expression vectors
• Table 2.9: Selection of suppliers of viral vectors for gene silencing
• Table 3.1: Suppliers of siRNA libraries for HT screening (3.1)
• Table 3.2: Commercial availability and coverage of shRNA libraries
• Table 3.3: Summary of the pros and cons of the different silencing reagents for RNAi screening
• Table 3.4: Advantages and disadvantages of RNAi knock-down in vivo
• Table 3.5: Key databases and algorithms for miRNA target prediction
• Table 3.6: Suppliers of miRNA mimics and inhibitors
• Table 4.1: Non-viral methods for siRNA delivery
• Table 5.1: RNAi therapeutics in, or close to, clinical development
• Table 5.2: RNAi therapeutic targets in neurological disease
• Table 5.3: Atugen' s pipeline of anticancer therapeutics
• Table 6.1: miRNA expression profiling in human cancers
• Table 7.1: Seminal patents in RNAi
• Table 7.2: Issues in Tuschl I vs Tuschl II
• Table 7.3: Licensees of Alnylam' s RNAi patents
• Table 7.4: Chemical modifications ? Sirna' s patent portfolio
• Table 7.5: Therapeutic targets for RNAi ? Sirna' s patent portfolio
• Table 8.1: Companies involved in RNAi technologies, A-B
• Table 8.2: Companies involved in RNAi technologies, C-G
• Table 8.3: Companies involved in RNAi technologies, I-O
• Table 8.4: Companies involved in RNAi technologies, P-Z
• Table 8.5: Sales forecasts for total RNAi market, 2006-2015
• Table 8.6: Sales forecasts for RNAi suppliers: synthesis and reagents, 2006 ? 2015
• Table 8.7: Pharma' s alliances for RNAi in R&D
• Table 8.8: Sales forecasts for delivery of RNAi therapeutics, 2006-2015
• Table 8.9: Costs associated with RNAi therapeutic development
• Table 8.10: Potential value of therapy areas targeted by RNAi therapeutics, 2005 & 2010
• Table 8.11: Sales forecasts for RNAi therapeutic drugs launched 2010-2015
• Table 8.12: Pharma' s alliances for RNAi in R&D