Therapeutic Opportunities of siRNAs and Antagomirs

Therapeutic Opportunities of siRNAs and Antagomirs

Tuesday, June 10, 2008

The New York Academy of Sciences

Presented By


Organizer: Thomas Tuschl, The Rockefeller University

The goal of this group is to provide a forum for scientists engaged in research into the biology, biochemistry, and applications of RNAi silencing to discuss advances in this exciting new field and to promote interinstitutional and interdisciplinary dialogue.




8:30-9:00 AM: Continental Breakfast

9:00-9:15 AM: Introductory Remarks

9:15-10:10 AM: Ingo Röhl

10:10-11:05 AM: Jost Seibler

11:05-11:35 AM: Coffee Break

11:35 AM-12:30 PM: Arthur M. Krieg

12:30-1:45 PM: Luncheon

1:45-2:40 PM: Jan Krützfeldt

2:40-3:35 PM: Philip S. Low

3:35-4:05 PM: Coffee Break

4:05-5:00 PM: John R. Lamb

5:00-6:00 PM: Reception



  Analytical Approaches to Determine Quality, Pharmacokinetic Properties and Biodistribution of siRNAs
Ingo Roehl, Roche Kulmbach GmbH, Germany

The development of appropriate analytical methods to measure siRNAs and their metabolites from biological material is an important prerequisite to successfully advance RNAi therapeutics from potent in vitro inhibitors to clinical drug candidates. The combination and improvement of instrumental analytical methods originally developed to address the quality of oligonucleotides, can serve to characterize improved stability and PK properties of siRNAs. A new HPLC-ESI-MS method allows the exact localization of nucleolytic cleavage sites in siRNA directly from biological fluids. The mass spectrometry results provide detailed information on the nucleases involved in individual cleavage events and thus enables an educated chemical stabilization strategy.

Furthermore a new and very robust HPLC based method is presented. The sample preparation procedure requires no enzymatic and RNA extraction or purification steps and therefore allows quantitative and highly reproducible recovery of analyte molecules over a broad concentration range. The assay allows the determination of siRNA concentration in tissue and plasma samples in the low ng/g range. Additionally the HPLC-based setup has a single nucleotide resolution and allows separation and identification of siRNA metabolites in PK and biodistribution studies.

Inducible shRNA Expression in Mice For Gene Function Analysis
Jost Seibler, TaconicArtemis GmbH, Germany

RNA interference through expression of short hairpin (sh)RNAs provides an efficient approach for gene function analysis in mouse genetics. Here we provide a generally applicable system for the temporal control of ubiquitous shRNA expression in mice. Depending on the dose of the inductor doxycycline, the knockdown efficiency reaches up to 90%. Our technology has been applied for more than 70 mouse models and therefore will lead to new insights into gene function and molecular disease mechanisms. Results from large number of mouse models demonstrate the high reliability of our inducible knockdown system. Moreover we have developed new approaches for the tissue-specific activation of shRNA vectors and the simultaneous expression of multiple shRNA transgenes from a single locus.

The Other Side of Antisense and siRNA – Immune Stimulation via Toll-like and RIG-I-like Receptors
Arthur M. Krieg, Pfizer

Antisense oligodeoxynucleotides (ODNs) and small interfering RNAs (siRNAs) are being developed as novel hybridization-based mRNA-specific therapeutics. Both antisense and siRNA agents can induce non-target-related biological effects including immune stimulation. The family of Toll-like receptors (TLRs) detect infections by binding highly conserved components of pathogens. A subset of TLRs, including TLR3, TLR7, TLR8, and TLR9, are expressed intracellularly within one or more endolysosomal compartments, and detect single-stranded CpG DNA (TLR9) and G,U-rich RNA (TLR7 and 8), or double-stranded RNA (TLR3). Another class of cytoplasmic nucleic acid receptors are the retinoic acid-inducible gene I (RIG-I)-like receptors (RLRs) with RIG-I responding to 5' terminal triphosphate modifications of double-stranded RNA. Signaling via these receptors results in innate immune stimulation and the production of type I interferon and/or pro-inflammatory cytokines. Specific chemical modifications of siRNAs and antisense ODNs can be used to suppress these immune effects, or the innate immune stimulation in combination with gene silencing may have useful therapeutic effects for cancer and infectious diseases. Moreover, TLR7/8 or RIG-I RNA agonists have shown some positive results in pre-clinical investigation as vaccine adjuvants and immune modulators in cancer and infectious diseases.

Characterization of miRNA Regulatory Networks using Antagomirs
Jan Krützfeldt, Institute of Molecular Systems Biology

MicroRNAs are regulators of many biological processes in development, differentiation, growth and metabolism. Their mode of action with a single microRNA potentially regulating hundreds of target genes poses unique challenges in studies elucidating microRNA function. We use algorithms to dissect microRNA regulatory networks in vivo applying antagomirs, a class of oligonucleotide inhibitors of microRNAs, as well as adenoviral-mediated overexpression of microRNAs. Messenger RNA profiling combined with bioinformatics analysis yields a unique opportunity to enhance our understanding of microRNA function. We are studying microRNA regulation in different models of metabolic disease and as an example a transcription factor-microRNA network of the liver will be presented.

Ligand-Mediated Targeting of siRNAs and Other Therapeutic Agents to Cancer Tissues and Sites of Inflammation
Philip S. Low, Erina Vlashi, Mini Thomas, Ligia Sega, and Yingqun Huang, Purdue

We have developed methods to deliver both low molecular weight and macromolecular drugs specifically to pathologic cells, thereby avoiding the collateral toxicity associated with drug uptake by healthy cells. In the case of cancer, we have exploited the strong upregulation of the folate receptor on malignant cells to target: i) siRNAs, ii) chemotherapeutic agents, iii) gene therapy vectors, iv) protein toxins, v) radioimaging agents, vi) nanoparticles, vii) liposomes with entrapped drugs, viii) immunotherapeutic agents, and ix) numerous types of imaging agents to tumor tissues by linking the various agents to the vitamin folic acid. Current clinical trials of five distinct folate-linked drugs demonstrate that the folate-targeting strategy holds great promise for significantly improving the magnitude and specificity of drug uptake by solid tumors. Folate-linked drugs must obviously extravasate and penetrate the tumor tissue before they can bind to FR-expressing cancer cells.

Inferring Causal Relationships between Genes and Disease in the Context of Molecular Networks
John R. Lamb, Merck

Common human diseases result from a complex interplay of genetic and environmental factors. To model this complexity we are taking the approach of sampling genetic, expression and phenotype variation in populations manifesting the disease of interest. Using this data we can infer causal relationships between genes and disease and integrate this with entirely data driven reconstruction of molecular networks. Emerging from this analysis are sub-regions of networks containing groups of co-regulated genes that collectively drive disease variation. Examples of individual targets and networks derived from mouse and human will be shown.