EXPLORING DIVERSE THERAPEUTIC INDICATIONS

8:35am Chairperson’s Remarks
John Zaia, M.D., Chairman, Department of Virology and Director of the General Clinical Research Center, Beckman Research Institute

8:40 tkRNAi-Based Therapeutics for Treatment and Prevention of HPV Related Cervical Cancer
Johannes Fruehauf M.D., Ph.D., Vice President, Research, Cequent Pharmaceuticals Inc.
Transkingdom RNA interference (tkRNAi) uses invasive nonpathogenic bacteria to direct RNA interference to mucosal surfaces in the gastrointestinal tract and other organs. Using this technology, Cequent Pharmaceuticals has an advanced-stage preclinical program to prevent polyp formation in Familial Adenomatous Polyposis (FAP), and is developing drug candidates for inflammatory bowel disease (IBD). Outside of the gastrointestinal tract, tkRNAi is being developed as a tool to treat dysplasia caused by infection with human papilloma virus (HPV), the leading cause for cervical cancer. E6 and E7 oncoproteins are silenced to restore functions of the tumor suppressor genes p53 and pRB. In this presentation, we introduce Cequent’s HPV-targeting program (CEQ400 series), discussing target selection, lead optimization, in vitro and in vivo proof of concept. With CEQ400, we are developing a tkRNAi -Based therapeutic to prevent cervical cancer in HPV infected patients using local treatment.

9:10 RNAi-Based Gene Therapy for Parkinson’s Disease and Related Neurodegenerative Synucleinopathies
Mohan Sapru, Ph.D., Assistant Professor, Neurobiology Program, CMRC, Feinberg School of Medicine, Northwestern University
Human α-synuclein overexpression and its toxic accumulation in neurons or glia are known to play key roles in the pathogenesis of Parkinson’s disease and other related neurodegenerative synucleinopathies. Silencing of the human α-synuclein gene by vector-based RNA interference (RNAi) is a promising therapeutic approach for synucleinopathies. Here, we report identification of a 21-nucleotide sequence in the coding region of human α-synuclein that constitutes an effective target for robust silencing by RNAi and demonstrate allele-specific silencing of the A53T mutant of human α-synuclein. Furthermore, we have developed a plasmid vector-based RNAi for silencing of human α-synuclein in vitro. Lastly, using a dual cassette lentivirus that co-expresses an α-synuclein-targeting small hairpin RNA (shRNA) and enhanced green fluorescent protein (EGFP) as a marker gene, we demonstrate effective silencing of endogenous human α-synuclein in vitro in the human dopaminergic cell line SH-SY5Y and also of experimentally expressed human a-synuclein in vivo in rat brain. Together, our results demonstrate potent silencing of human α-synuclein expression in vitro and in vivo by viral vector-based RNAi and provide the tools for developing effective gene silencing therapeutics for synucleinopathies, including Parkinson’s disease.

9:40 Delivery of shRNA and Other Anti-HIV RNAs by Autologous Transplantation of Lentivirus-Transduced CD34+ cells: A Feasibility Trial
John Zaia, M.D., Chairman, Department of Virology and Director of the General Clinical Research Center, Beckman Research Institute
A lentivirus encoding three anti-HIV RNAs was used for the first time in a clinical trial. The vector encoded an shRNA targeted to tat/rev, a TAR decoy, and a CCR5-specific ribozyme. The problems and approaches to design such a vector, the large-scale packaging of a lentivirus encoding inhibitory packaging elements, and the issue of manufacture and release testing of the final cell product will be discussed. The study is enrolling subjects, and two AIDS lymphoma patients have been treated to date. The study status will be updated at the time of the presentation.

10:10 Networking Coffee Break in the Exhibit Hall

10:55 Development of Clinically Applicable RNAi Therapy for Liver Fibrosis
Jian Wu, M.D., Ph.D., Associate Professor, Department of Internal Medicine, Transplant Research Program, University of California, Davis Medical Center
Liver fibrosis is a progressive disorder developed concurrently during or after chronic liver injury. To develop a clinically applicable RNAi strategy to treat this disease requires that the vector has liver-preferential delivery, and that the overexpression of shRNA will be sustained for a prolonged time period and is controllable. We are in the process to develop a lentiviral vector in which the overexpression of shRNA against transforming growth factor beta receptor II (TGFbRII) is inducible with a Tet-responsive element, and is selective with a hepatic stellate cell (HSC)-specific promoter. Thus, the vector will selectively express high levels of shRNA against TGFbRII, the receptor of a most potent fibrogenic cytokine, in HSC, a major cell type which is responsible for overproduction of extracellular matix components.

11:25 siRNA Therapeutics for Treatment of Pathological Ocular Angiogenesis and for Retinal Neuroprotection
Evgenia Alpert, M.D., Ph.D., Project Director, Eye, Ear and CNS Disorders, Research Division, Quark Pharmaceuticals Inc.
siRNA targeting Quark’s proprietary gene RTP801 has been extensively studied in the model of laser-induced choroid neovascularization in mice. Its intravitreal injection resulted in significant and dose-dependent reductions of CNV volume, neovasculature leakage and inflammatory cell infiltration into choroid. Anti-angiogenic effect of RTP801 is likely VEGF-independent and siRTP801 showed at least additive effect when administered together with VEGF inhibitors. The drug has currently completed Phase 1 clinical trials. Additional siRNA therapeutics currently under pre-clinical development for ophthalmologic indications target two different genes. Each of these siRNAs confers significant protection of retinal ganglion cells in acute retinal neurodegeneration disease models in vivo.

11:55 Therapeutic RNAi in the Vascular Endothelium with Liposomal siRNA (AtuPLEX)
Ansgar Santel, Ph.D., Senior Scientist, Silence Therapeutics AG
Pathological angiogenesis and vascular dysfunction contribute to several different disorders such as cancer, arthritis, atherosclerosis and inflammatory processes. Therefore, an RNAi-mediated modulation of the vascular endothelium in vivo would offer many opportunities for developing new therapeutic interventions. The AtuPLEX, a liposomal formulation of siRNAs (AtuRNAi) enables RNAi-mediated gene silencing in the vasculature after systemic administration. Silence’s current efforts in employing the AtuPLEX for therapeutic purposes will be discussed, with particular focus on pulmonary infection and cancer diseases. Pre-clinical research data will be presented, including an update on our current drug candidate Atu027, demonstrating the broad therapeutic applicability of the AtuPLEX.

12:25pm Luncheon Workshop (Sponsorship Available) or Lunch on Your Own

ADDRESSING SAFETY AND MANUFACTURING CONCERNS

1:55 Chairperson’s Remarks
Mark Behlke M.D., Ph.D., Chief Scientific Officer, Integrated DNA Technologies

2:00 Safety Assessment of siRNA-Based Therapeutics
Thomas Singer D.A.B.T., Global Head of Non-Clinical Safety, F. Hoffmann-La Roche AG
siRNA-Based therapeutics offer the unique opportunity to target virtually any gene of the human genome leading to a significant extension of therapeutic opportunities. However, safety assessment turns out to be more complex as with classical drugs because the delivery vehicle and the double stranded RNA trigger independent responses such as immune system or transcriptome interference. Strategies to address these interactions will be presented together with a panel of standard assays we consider important and relevant for drug safety of this novel class of medicines.

2:30 Human Primary Hepatocyte/PBMC Co-Culture for Examination of Immune Modulation by RNA-Based Therapeutics
Eric Tien Ph.D., Senior Scientist, Systems Biology, Pfizer
A major safety issue surrounding therapeutic RNAi development is immune modulation in response to RNA by toll-like receptors (TLR). Human peripheral blood mononuclear cells have been widely used to assess immune modulatory activity of nucleic acids, including RNA. We developed a human primary cell co-culture system to examine how cytokine secretion by PBMCs in response to RNA affects hepatocyte functionality. We observed hepatic transporter activity was inversely proportional to the immune stimulatory activity of RNAs. Our results provide a potential mechanism for cholestatic effects of TLR ligands including LPS and RNA, and support co-culture models in early risk assessment of RNA-Based therapeutics.

3:00 Networking Ice Cream Refreshment Break in the Exhibit Hall (Last Chance for Viewing)

3:40 Manufacturing, Safety and Efficacy of SNALP Formulated siRNA
Ian MacLachlan, Ph.D., Chief Scientific Officer, Tekmira Pharmaceuticals, Inc.
The ideal siRNA delivery system will (i) be safe and well tolerated upon administration; (ii) have the appropriate pharmacokinetic attributes to ensure delivery to disseminated disease sites and (iii) be stable upon manufacture facilitating production at commercial scale. Stabilized nucleic acid lipid particles (SNALP) are a modular lipid nanoparticle delivery platform developed for the delivery of siRNA. SNALP pharmacology can be modulated in a predictable manner by manipulating the structure of individual SNALP lipids. SNALP mediated RNA interference, using siRNA, has been confirmed in several preclinical models of oncology, infectious and metabolic disease. SNALP formulated siRNA are expected to enter Phase I clinical trials in mid-2009.

4:10 Manufacturing RNA Duplexes for Research and Pre-Clinical Studies
Mark Behlke M.D., Ph.D., Chief Scientific Officer, Integrated DNA Technologies
A variety of chemistries and purification approaches can be used to manufacture synthetic RNA oligonucleotides for use in RNAi research. All of these methods can result in high quality oligonucleotides, however scale up to high throughput or large scale synthesis is more challenging. A description of quality control issues from a manufacturing perspective will be discussed using synthesis of Dicer-Substrate siRNAs as an example.

4:40 Analytical Characterization of siRNA Duplexes in a cGMP Manufacturing Environment
Todd Kreutzian, Director of Analytical Development, Nucleic Acid Solutions Division, Agilent Technologies
There has recently been increased therapeutic interest in DNA and RNA duplexes. While manufacturing methods have advanced for individual strand synthesis and annealing, analytical methods for characterization of these material has often relied on legacy SEC or GPC approaches. These methods tend to significantly overestimate the purity of duplexes based on the levels that could be achieved theoretically as calculated from the known purity of the individual single strands. In addition, they lack the resolving power to adequately separate and therefore quantify mismatched duplexes. Anion exchange HPLC exhibits better resolution compared to size separations, but is not compatible with mass spectrometry; limiting the ability to gather critical product and impurity information during development. Reversed phase ion-pair HPLC / HPLC-MS methods have been developed that exhibit superior resolution compared with ion exchange HPLC and SEC/GPC. Ion-pair HPLC using mass spec detection may provide purity results that are more closely aligned with theoretical values and possible identification of mismatched duplex species. Presented are comparisons of these analytical methods and some interesting findings on the impurity duplexing tendency of early sequence failures versus late sequence failures.

5:10 Close of RNAi Therapeutics Conference