1104 argonaute 2 is a key limiting factor for therapeutic RNAi in the liver

1104 Argonaute 2 Is a Key Limiting Factor for Therapeutic RNAi in the Liver triggers than our I" first generation trigger, HBVU6#2 Khovorova et al and Schwarz et al demonstrated that an siRNA''''s intern[.]

triggers than our I" first generation trigger, HBVU6#2 Khovorova et al and Schwarz et al demonstrated that an siRNA's internalthermodynamic stability profile (ISP) determines whether the desired antisense strand is efficiently incorporated into the RNA Induced SilencingComplex (RISC) Weused the webtool, SFOLD to identify HBV RNAi triggers most closely conforming to the ISPdescribed by Khovorova et al Wethen incorporated GU base pairs into the sense strand that altered the thermodynamic profile to more closely match the consensus These triggers were designed to conform to reported sequence preferences for efficient RNAi described by Reynolds et at Wealso selected triggers that were predicted by SFOLD to target regions in HBV RNAs that are accessible to hybridization RNAi triggers embedded in endogenous miRNA scaffolds are more efficiently processed into mature siRNAs than shRNAs Therefore, we expressed our HBV RNAi triggers in the context ofthe endogenous miRNA, miR30.This will also allow expression using liver-specific and inducible promoters 14 out of 15 of the rationally designed HBV RNAi triggers were more potent in cultured cells than our I" generation construct, HBVU6#2 Northern blots indicated that our rationally designed RNAi triggers favored incorporation of the desired antisense guide strand into the RISC complex However,our data also suggest that other downstream parameters influencesilencing efficiency These results demonstrate that rational approaches can be used to reliably design more potent RNAi triggers Studies with these optimized triggers in mice will be discussed Because ofthe general nature of these approaches, they could be adapted to the treatment of diverse infections and diseases 1102 Novel "Bifunctional" shRNA Knockdown Therapeutics to Individualized Cancer Targets John Nemunaitis.P" Phillip B Maples.P Donald Rao,' Joseph Kuhn,' Yuqiao Shen.! Alex W Tong.t-' Chris Jay" ,2 Padmasini 2,3,~ Kumar,' Neil Senzer.I [Cancer Research, Mary Crowley Cancer Research Centers , Dallas, TX; 2Cancer Research, Murex Pharmaceuticals, lnc , Dallas, TX; JCancer Research, Baylor Sammons Cancer Center; Dallas, TX; 'Cancer Research, Texas Oncology; I~A , Dallas, TX We have created a novel process of identifying cancer specific and, potentially critical, molecular targets in individual patients and have constructed corresponding RNA interference therapeutics to test clinical impact Malignant and non malignant tissue from 81 cancer patients were harvested Differentially overexpressed protein and correlated mRNA signals were determined (mRNAArray, UTSWMC, Dallas, TX and 2D DIGE/MS; Applied Biomics, San Francisco, CA) in 32 of these patients Gene expression pathway modeling(GNS, Boston, MA) of prioritizedproteinswith pro-cancer function overexpressed in cancer allowed for determination of intra pathway protein connectivity in IS patients Novel "bifunctional" shRNA constructs, which demonstrate both cleavage dependent and cleavage independent RISC (RNA induced silencing complex) mediated inhibition of the targeted homologous mRNA, were constructed and tested The "bifunctional" shRNA has produced up to a 260 percent enhanced knockdown of the target protein (compared to traditional siRNA knockdown to the same sequence at optimal dose), at 48 hours after clonal cancer cell exposure Furthermore, consequent functional modulation (e.g., apoptosis) was observed Duration of target knockdown, measured by sequential Western blot and alkaline phosphatase reporter assay, demonstrated a further temporal advantage of bifunctional shRNA over siRNA (at optimal doses) beyond 48 hours Subsequent treatment of malignant clonal cells with known high expression ofthe target protein confirmed increased cell death over time following exposure to the "bifunctional" shRNA A clinical grade plasmid containing a unique promoter for tumor specificcontrolled shRNAexpression was engineered.Animal efficacy and toxicology testing are underway using the target vector Molecular Therapy Volume 15, Supplement I May 2007 © Th e American Socie ty o f G ene Th erap y Copyright packaged in a tumor antigen targeting immunoliposome nanoparticle for delivery The clinicallND justifying patient treatment is in process Update of results will be presented 1103 Using Let-7 microRNA To Inhibit Pancreatic Cancer Cells Proliferation Laurie Parmentier,' Jerome Torrisani,' Barbara Bournet,' Anny Souque,' Michele Bouisson,' Jean Escourrou,' P Senesse,' Marc Barthet,"N Lesavre,' Philippe Ruszniewski,' Dermot O'Toole,' A Aubert,' Pascal Hammel,' Philippe Levy,' Janick Selves," Lucien Pradayrol,I Louis Buseail, I Pierre Cordclicr.' IINSERM U858, 12MR, Toulouse, France; 2Service de Gastroenterologie, CflU Rangueil, Toulouse, France; 'Service de Gastroenterologie, Val d'Aurelle, Montpelliet; France; 'Service de Gastroenterologie, CHU Nord et CIG, Marseille, France; ' Service de Gastroenterologie, Hopital Seal/jon, Clichy; France; 61NSERM U563, CHU Purpan, Toulouse, France Background; pancreatic ductal carcinoma is the fifth leading cause of cancer related deaths in Westerncountries, with an increasing incidence over the last 40 years So far, no effective therapies have been established to alleviate this devasting and often fatal endstage condition Thus, there is an urgent need for the development of new modalities to improve diagnosis and treatment of pancreatic cancer MicroRNAs (miR) are small non-coding transcripts that regulate gene expression by inhibiting translation Emerging studies suggest that many miRNAs may participate in human disease, including oncogenesis The aim of present work was to establish the pattern of expression of miR known to potentiate or to inhibit oncogenesis in PC-derived cell lines, tumors and fine needle aspiration (FNA) Methods: Total RNA was purified from PC-derived cell lines, tumors and FNA RNA collected by aspiration was amplified using Full Spectrum amplification kit As controls, we used RNA purified from normal, adjacent pancreas to pancreatic cancer miR levels were ascertained by Taqman RT-PCR Results: using Taqman-based RT-PCR, we identified a global down regulation of anti-oncogenicmiR (miR-21, miR-22I, miR-222,miR-16, miR-15a, miR 17-5-p, Let-7a) in pancreatic cancer samples, when oncogenic miR were elevated (miR-211, mir-30I) Let-7 miR down regulation was further confirmed by Northern blotting and in situ RT-PCR on pancreatic adenocarcinoma tissue micro array Eventually, we identified a global down expression of let-7 miR family members in 30 FNA of pancreatic cancer tumors Because ki-RAS oncogene is a validated target of Let-7 miR, we transfected pancreatic cancerderived cells with Let-? miR We observed a dramatic decrease in PC-derived cells proliferation, with a eoncomittant inhibition ofkiRAS protein expression Conclusion: a better understanding of the molecular mechanisms involved in pancreatic cancer oncogenesis could lead to the development of new molecular markers for either early diagnosis or targeted therapy Here we show that anti-oncogenic miR expression, such as let-7, is strongly down regulated in pancreatic cancer samples Taken together, this study describes the potential clinical and therapeutic interest of delivering Let-7 for treating pancreatic cancer 1104 Argonaute-2 Is a Key Limiting Factor for Therapeutic RNAi in the Liver Dirk Grimm, Lora Wang, Joyce S Lee, Theresa A Storm, Mark A Kay I Pediatrics and Genetics, Stanford University, Stanford, CA We recently reported efficient and persistent suppression of Hepatitis B virus (HBV) in mice using double-stranded AAV-8 vectors expressing anti-HBV shRNAs A striking finding was that shRNA over-expression from a robust U6 promoter frequently caused hepatocellular toxicity, rapid organ failure and lethality S421 Comprehensive analyses of various shRNAs and targets implied the cause was saturation of the endogenous RNA i pathway, especially the sh/miRNA transporter Exportin-5 Here, we show that compared to U6, shRNA expression from weaker HI or 7SK promoters typically induces delayed but more persistent RNAi in mice This substantiates the correlation of intra-cellular shRNA levels and cytotoxicity We then tried to actively rescue mice from shRNA toxicity, by co-expressing Exportin-5 from liver-specific AAVs together with vectors transcribing high levels of shRNAs We observed a modest increase in RNAi efficacy, but surprisingly, it was only transient and the toxicity phenotype actually manifested more rapidly This suggested that a second factor downstream in the RNAi pathway had become limiting due to abundant Exportin-5 expression To identify this factor, we co-transfeeted cells and mice with robust shRNA constructs and plasm ids expressing key elements of the mammalian RNAi pathway, including essential components of RISC While Drosha and TRBP over-expression had no effect, Dicer co-transfection actually reduced shRNA activity Argonaute-I, -3 and -4 had an even stronger dominant -negative effect, suggesting competition with the active RISC complex in the cell Remarkably, over-expression ofArgonaute-2 (the "Slicer" enzyme in mammalian RISC which cleaves the target mRNA) enhanced RNAi activity by at least 5-fold in vitro and in vivo, even with our most efficient shRNAs The increase was greater than with Exportin-S, implying that Argonaute-2 is the main limiting factor in the RNAi pathway in mammalian cells and in murine liver To study the long-term effects, we infused AAV-8 vectors expressing Argonaute-2 into mice transgenic for human alpha-l-antitrypsin (hAAT), together with a robust U6-anti-hAAT shRNA Control mice expressing only the shRNA showed a transient knockdown that peaked at week and was entirely lost at week 4, due to liver toxicity and regeneration In striking contrast, Argonaute-2 co-expressing mice continued to show a stable 20-fold hAAT knockdown for over I month Mice expressing Exportin-5 together with Argonaute-2 showed an even further 2-fold RNAi increase Our data strongly suggest that therapeutic RNAi in the liver will be restricted by at least two endogenous factors Because the more limiting, Argonaute-2, is utilized by all conventional RNAi triggers (shRNAs, miRNAs or siRNAs), our findings have a broad and general impact Critical to the success of therapeutic liver RNAi will be to avoid Exportin/Argonaute saturation, either by tightly controlling intra-cellular hairpin or siRNA concentrations Alternatively, our data suggest that a promising novel approach is to co-express the limiting factors together with the shRNA Such combinatorial strategies could broaden the therapeutic index of current RNAi triggers and thus substantially enhance the safety and efficacy of the approach 1105 Prevention and Reversal of Autoimmune Diabetes by Microsphere-Formulated Antisense Oligonucleotiudes Targeting Costimulation Karen Nylander,' 10 Harnaha,' Robert Lakomy,' Alexis Styche,' Kimberly Gillis,' Larry Brown,' Massimo Trucco,' Nick Gi- annoukakis.' I Diabetes Institute University of Pittsburgh School ofMedicine Pittsburgh, PA; 2Ep ic Therapeutics Baxter Health Care Corporation Norwood, MA Prevention and reversal ofautoimmune diabetes by microsphereformulated antisense oligonucleotiudes targeting costimulation Type I diabetes mellitus is a disorder ofglucose homeostasis caused by a chronic autoimmune inflammation of the pancreatic islets of Langerhans The inflammation compels migratory antigen presenting cells, dendritic cells most prominently, to acquire beta cellresident antigens derived from apoptotic and/or necrotic beta cells The migratory dendritic cells then undergo an intrinsic "maturation" program which renders them capable ofactivating T-cells (including S422 autoreactive , beta cell-specific T-cells) as they accumulate inside the draining pancreatic lymph nodes We have previously shown that administration of dendritic cells with low-level expression of CD40, COSO and CDS6 costimulatory molecules into the genetically-diabetic non-obese diabetic (NOD) mouse can considerably delay and prevent the onset of disease and can reverse new-onset disease Considerable logistics to generate these dendritic cell embodiments, necessitate a means to stabilise immaturity directly in vivo Microparticle carriers can direct DC to the administration site and once phagocytosed, the contents can shape the DC unctional phenotype We have adapted PROMAXXTM microspheres to formulate antisense oligonucleotides previously shown to render autologous dendritic cells diabetes-suppressive This formulation, when injected subcutaneously prevented type I diabetes in the nonobese diabetic (NOD) mouse strain and most importantly, exhibited a capacity to reverse new-onset clinical hyperglycemia, suggesting reversal of new onset disease Mechanistically, we demonstrate that the formulation augments CD25+ Foxp3+ T regulatory cells which confer suppressive activity in vitro to NOD-derived pancreatic beta cell antigen without compromising global immune response to alloantigen and nominal antigen Furthermore, T-cells from diabetes-free recipients of the microspheres are capable of suppressing adoptive transfer of disease by diabetogenic splenocytes into secondary immunodeficient recipients We believe that these data form the foundation upon which a bona fide type I diabetes negative vaccine can be developed that is logistically-simple to produce and rapidly scalable to population levels STEM CELLS - BASIC SCIENCE SESSION 1106 High Proteasome Activity Is a Common Feature among Stem Cells of Different Lineages and Restricts Lentiviral Gene Transfer Francesca R Santoni de Sio,I.} Paolo Cascio ,' Angela Gritti,' Margherita Ncri ,' Maurilio Sampaolesi,' Silvia Colleoni,' Cesare Galli,' Luigi Naldini.':' 'Telethon Institute for Gene Therapy (TIGET), San Raffaele Scientific Institute, Milan, Italy; 21ljta-Salute University, San Raffaele Scientific Institute, Milan, Italy; 'Schoot of lIeterinmy Medicine, University ofTurin Turin, Italy; 'Stem Cell Research Institute, San Raffaele Scientific Institute, Milan, Italy; sReproductive Technologies Laboratory; Istituto Sperlmentale L Spallanzani, Cremona, Italy An emerging field of studies has demonstrated that mammalian cells express factors that restrict retrovirus and retrovirus-derived vector infection Although Hematopoietic Stem Cells (HSC) can be transduced by HIV-derived lentiviral vectors (LV) in short ex vivo culture , they display low permissiveness to the vector, requiring cytokine stimulation and high vector doses to reach high-frequency transduction We demonstrated that the use of proteasome inhibitors during transduction dramatically enhanced HSC gene transfer, allowing reaching very high levels of vector integration in their progeny in vivo, without apparent detrimental effect on their engraftment ability Thus , LV are effectively restricted by the activity of this proteolytic complex, Here, we investigated the cell type specificity and the molecular mechanism ofthis restriction Surprisingly, among a wide panel of primal)' and continuous cell cultures, protcasomc restriction of LV was present only and in all the stem cells tested , including human and murine HSC, human embryonic stem cells (ESC), murine mesenchimal stem cells (MSC) and murine neural stem cells (NSC) Remarkably, in cells differentiated from HSC (myeloid and erythroid lineages) , MSC (smooth muscle cells) and NSC (glia and neurons) proteasome restriction was lost Thus , the observed restriction seems to be a characteristic feature of stem cells in their primitive state We biochemically tested the proteaMolecular Therapy Yofume 15 Supplement I, \by 2007 Copyright © '111C American Societyo f Gene TIICr.lpr ... limiting factor in the RNAi pathway in mammalian cells and in murine liver To study the long-term effects, we infused AAV-8 vectors expressing Argonaute- 2 into mice transgenic for human alpha-l-antitrypsin... showed an even further 2- fold RNAi increase Our data strongly suggest that therapeutic RNAi in the liver will be restricted by at least two endogenous factors Because the more limiting, Argonaute- 2, ... enhanced RNAi activity by at least 5-fold in vitro and in vivo, even with our most efficient shRNAs The increase was greater than with Exportin-S, implying that Argonaute- 2 is the main limiting