698. Mutation-Independent RNAi for Rhodopsin-Linked Autosomal Dominant Retinitis Pigmentosa

vectors had on the processing and function of artificial miRNAs designed to mimic endogenous stem loops. Northern blot analysis showed that shRNAs markedly inhibited processing of artificial miRNAs, while miRNA shuttles had relatively no effect even at much higher doses. Similarly, shRNAs at low doses drastically reduced the level of gene silencing mediated by artificial miRNAs ; whereas , miRNA shuttles at high doses only caused slight inhibition. Notably, silencing efficacies of miRNA shuttles (high dose) and shRNAs (low dose) were indistinguishable. These results suggest that shRNA-based vectors saturate cellular RNAi machinery substantially more than miRNA shuttles. Hence, miRNA shuttles may provide a safer approach to therapeutic RNAi in vivo. In ongoing work, we are assessing the effects that shRNAs and miRNA shuttles have on endogenous miRNA levels and function in vivo. This work is supported by the NIH and the HOF.

696. Efficient Knock-Down of Prion Protein by Lentivectors Expressing Short Hairpin RNAs Andreas Hofmann,' Sabina Eigcnbrod.' Saba Al-Khadra,' Markus Moser,' Uwe Bertsch ,' Hans Kretzschmar;' Alexander Pfeifer.' 'Institute for Pharmacology and Toxicology, University ofBonn , Bonn. Germany; 2Cenfre for Neuropathology and Prion Research. Ludwig Maximilians University ofMunich. Munich, Germany; 'Molecular Medicine, MPlfor Biochemistry, Munich. Germany. Prion diseases or transmissible spongiforme encephalopathies (TSEs) are fatal neurodegenerative diseases. They are characterized by the accumulation of PrpSc, which is the protease-resistant and infectious form of the normal cellular prion protein (Prl"). Since Prpc-deficient (Prnp %) mice are resistant to prion disease, targeting of Prpc by RNA interference (RNAi) is a promising approach for the treatment ofTSEs. Lentiviral vectors stably integrate into the host genome and allow for long-term transcription of siRNAs. To induce RNAi in vitro and in vivo, SiRNA design: lentiviral vectors (LVshPrP) were designed to express short hairpin RNAs (shRNAs) directed against murine Prpc under the control of the H I RNA polymerase III promoter. LVshPrP also carries EGFP as a reporter gene to identify infected cells and tissues. Transduetion of murine N2a neuroblastoma cells with LVshPrP (MOI=IO) resulted in a 97% knock-down of Prpc. In contrast, lentivectors carrying scrambled shRNA had no significant effect on Prpc expression. Next, we analysed the effect of LVshPrP in primary neuronal cells. LVshPrP transduced granule cells demonstrated a 85% reduction of Prpc expression within 3 days after infection. To analyse the efficacy of LVshPrP in vivo , we generated lentiviral transgenic mouse lines by infection of embryonic stem (ES) cells. In the brain of ES-cell derived chimeric mice (80-90% coat colour chimerism), a reduction of Prpc levels of up to 71% was observed. Most importantly, mice with high degree of chimerism (> 65%) showed significantly prolonged survival times after scrapie infection. Taken together, lentiviral shRNA vectors are versatile tools to efficiently silence Prpc expression in vitro and in vivo.

697. Therapeutic Gene Knockdown with Wrapped Liposome-Mediated Systemic siRNA Delivery Nobuhiro Yagi,' Ichiro Manabe .? Hidenori Takahashi.? Jongheon Kim.' Katsuhito Fujiu.' Yumiko Oishi.' Masahiro Yamauchi,' Noboru Aoki , I Yasuhiro Tamaki.! Ryozo Nagai.! 'Pharmaceutical Research Center; Kyowa Hakko Kogyo Co., Ltd., Nagaizumi-cho, Sunto-gun, Shizuoka, Japan; 2Graduate School ofMedicine, The University of Tokyo, 7-3-1 Hongo Bunkyo-ku, Tokyo, Japan. RNAi is a prom ising technology that would lead to novel therapeutics against various diseases. To enable systemic delivery and Molecular Therapy Volume 15.Supplement 1. ~by Cop yright © The Americ...m Society o r Gene Therapy

2007

therapeutic gene knock down of siRNA in vivo, we developed a novel nanoparticle technology, the Wrapped Liposome (WL). WL was designed to extend the half-life of siRNA in systemic circulation and deliver siRNA to the areas of vascular hyperpermeability, WL consists of lipid molecule and siRNA, and has unique structural characteristics including a small and homogeneous diameter, the lipid-enveloped architecture, and the surface coverage of hydrophilic polymers. WL was translucent in solution, and their size distribution was Gaussian with an average diameter of approximately 100 nm. siRNA incorporated into WL exhibited enzymatic resistant to RNaseA, and plasma half-life of WL was determined to be approximately 18 h. Wrapped liposomal fluorescence-labeled siRNA exhibited gradual accumulation into subctaneously implanted xenograft tumors and laser-induced model of choroidal neovascularization (CNV) spontaneously, and they culminated 24 h after single injection to tail vein. siRNAs against KLF5, BcI-2 and Flt-l were incorporated into WLs and evaluated therapeutic effects. Transcription factor KLF5 plays important role in angiogenesis and tissue remodeling in the cardiovascular system. KLF5 heterozygous knockout mice exhibited much reduced tumor angiogenesis and progression in a tumor implantation model, suggesting that a suppression of KLF5 by siRNA would lead to significant inhibition of angiogenesis. We found that only KLF5siRNA incorporated into WLs exhibited significant antitumor activity (inhibited tumor growth approx. 70%) among all the formation administered; neither WLs containing control scrambled-siRNA nor saline containing KLF5siRNA affected tumor growth. Only KLF5siRNAlWL inhibited KLF5 expression significantly at the mRNA and protein levels, while all the control did not. Angiogeneis was significantlly reduced in tumors in mice treated with KLF5siRNA/WL. On the other hand, there were no significant differences in body weight and no histological abnormalities were detected in the liver, lungs and spleen. To further evaluate the potential of WL as the systemic delivery system for siRNA, we generated the siRNA against BcI-2, which is known to be involved in apoptosis. In the tumor xenograft model, bcl-2siRNAlWL also significantly inhibited the growth of solid tumor. Furthermore, we tested opthalmological applications of WL. The vascular endothelial growth factor (VEGF) signaling pathway plays an important role for CNV. Clinical trials against retinal neovasucularization is now conducted by intraviterous administration ofsiRNA. Systemic administration ofFlt-lsiRNA/WL resulted in a significant reduction of'neovascularization in the mice CNV model. Results of the present study clearly demonstrate that WL enables systemic delivery ofsiRNA to the target sites and induce the therapeutic gene knockdown.

698. Mutation-Independent RNAi for Rhodopsin-Linked Autosomal Dominant Retinitis Pigmentosa Naomi Chaddcrton,' Arpad Palfi,' Mary O'ReiIlY,l Sophia Millington-Ward, I Marius Ader, 1 Gcaroid Tuohy,' Peter Humphries,' Paul F. Kenna , '.2 G. Jane Farrar.' 'Genetics, Trinity College Dublin. Dublin, Ireland; ]Research Foundation, Royal Victoria Eye and Ear Hospital. Dublin. Ireland. Retinitis Pigmentosa (RP), the most prevelant cause ofregistered blindness in people of working age in the developed world , represents a family of genetically heterogeneous inherited diseases . The development oftherapies for autosomal dominant RP (adRP) poses significant challenges given the inter- and intragenie heterogenei ty. Mutations in the rhodopsin (RHO) gene account for the largest proportion of adRP cases , with over 100 mutations identified to date. To overcome mutational heterogeneity we have proposed a suppression and replacement therapeutic strategy. We have recent ly explored the suppression aspect ofthis approach using short hairpin S267

RNAs (shRNAs) targeting human RHO in both cell culture and retinal explants. In the current study we explored whetherefficient RNAi-based suppressionof humanRHOcould be achieved in vivo. We have generated an adeno-associated virus (AAV) expressing the most efficient RHO targeting shRNA (identified in previous studies) together with an EGFPgene to monitor viral transduction, AAV2/5.shRHO-EGFP. Transgenicadult mice expressinga human rhodopsin gene (NHR+I· rho") were subretinally injected with 3ul AAV2/5.shRI-IO-EGFP inone eye, whilstthe otherwas injected with a non-targeting control AAV (AAV2/5.shNT-EGFP). Retinas were harvestedtwo weekspost-injection,dissociated and EGFP-positive cells collected by PACS. ExpressionofshRHO was confirmed by RNase protection (n=2). Analysis of human RHO mRNA levels by quantitativereal-time IU-PCR showed significantsuppression, greater than 85%, in those retinas receivingAAV2/5.shRHO-EGFP (n=6). This was verified at the protein level by rhodopsin immunohistochemistry (n=4). Suppressionalone is unlikely to provide a long term therapy due to the necessity of a functional replacement RHO gene. However, the data presented provide c1car evidence of in vivo RNAi-mediated suppression in photoreceptorcells and validate the functionality of the suppression element of this mutation-independent therapeuticapproach.

699. Allele-Specific Silencing of Mutant Huntingtin for Huntington's Disease Therapy

Alex Mas-Monteys,' Scott Q. Harper,' Brian L. Gilmore; Patrick Staber,' Chris Schaffer,' Barry Pollsky.! Chandra Vargesse.? Beverly L. Davidson. ' 'Internal Medicine, University ofIowa. Iowa City, IA; 2Biology, Sirna Therapeutics, Boulder, Co. Huntington's disease (HD) is a dominant genetic neurodegenerative disease. Studies in a tetracycline-responsive mouse model showed that continuous expression of the mutant allele is required forthedevelopment of behavioral and pathological symptoms; when mutant huntingtin expression was turned off, disease phenotypes resolved. More recently, gene therapy approaches using RNA interference(RNAi) in HD mousemodelssuccessfullypreventedHD phenotypes. Because we do not know if reduction of both normal and mutanthuntingtinalleles will be safely tolerated in humans,we investigated an allele-specific RNAiapproach for HD.Wedeveloped novel constructs to test if certain siRNAs could target the mutant allele preferentially. Briefly, we generated plasmids expressing full-length wild type or mutant huntingtin,each possessingdistinct epitopetags and containingdifferentexpressioncassettesfor renilla (WT htt) or firefly (mutant htt) luciferaseto normalize transfection efficiencies. Using this design we assessed simultaneously allele specificity. Short interfering RNAs (siRNAs) targeting full length htt were generated and co-transfected into cells with the full length plasmids. Q-PCR and western blot showed that some siRNAs silencedthe mutantallele by 40% butthe wild type huntingtin by only 6%. Together, our data indicatethat differences in mRNAstructure may permit allele specificsilencing in HD therapy.

700. Development of RNA Aptamers for Human Toll-Like Receptor 2

HyoungreaYoo, Hoon YoungKong, Han-Na Lee, Young-Ju Lee, Seong-Wook Lee, Jonghoe Byun. 'Department ofMolecular Biology, BK2I Graduate Program for RNA Biology, Institute ofNanosensor and Biotechnology, Dankook University; Seoul, Republic ofKorea.

Currently, there is a growing interest in the biology of toll-like receptors (TLRs). These pattern-recognition receptors (PRRs), expressed on various immune and non-immune cell types, playa crucial role in the innate immune response to microbial infection. S268

Their activation by various ligands triggers a signalling cascade leadingto cytokineproduction and initiationof an adaptiveimmune response. However, despite these importantaspects of'Tl.Rs, there arc few therapeutic agents directed against these receptors for the treatment of the diseases. Here, we focused on the developmentof RNA-based aptamers that can specifically bind to and modulate the function ofTLR2. To achieve this goal, we generateda soluble formof humanTLR2 (sTLR2)lackingthe putativeintracellularand transmembrane domains. This truncatedTLR2 protein(Meti-Arg'") was then subjectedto SELEX(Systematic Evolutionof Ligandsby Exponential enrichment) procedure to obtainRNAaptamersthat can selectively bind to the eetodomains ofTLR2. Isolation of the best performing aptameris currentlyunderway and subsequentcharacterizationstudies includingSurface Plasmon Resonance analysis will be presented. Since TLRs recognize specific molecular patterns of microbial components and their stimulation relates to both innate and adaptiveimmunity, TLR2aptamersmightprovidenoveloptions in the treatment of many infectiousdiseases.

701. Restoration of Dystrophin Expression in the Diaphragm of the Dystrophic mdx Mice by Polymer-Tagged Morpholino Antisense Oligonucleotides

Bo WU,I Pei Juan LU,I Jignya Ashar,' Ehsan Bcnrashid,' Elizabeth Kcrarnaris,' YongFu Li,2 Paul A. Morcos," Qi Long Lu. 1 lMcColI-Lockwood Laboratoryfor Muscular Dystrophy Research, Cannon Research Center; Carolinas Healthcare System, Charlotte, NC; 2Gelle Tools, LLC, Philomath, OR. Duehenneand Becker musculardystrophiesarc allelic disorders arising from mutations in the dystrophingene. Duchennemuscular dystrophy (DMD) is characterised by nonsense or frame-shifting mutations in the dystrophin gene that result in an absence of functional protein, dystrophin, while Becker muscular dystrophy is usually caused by in-frame deletions resulting in shortened yet partially functional dystrophin. The application of antisense oligonucleotides (AOs) is a promising strategy for the treatment of DMD. This treatment removes specific exons from the dystrophin mRNA transcript with AOs, thus altering pre-mRNA processing to restore the mRNA reading frame and to express shortened but functional Becker-like protein. Local and systemic deliveries of phosphodiarnidate morpholino oligonucleotides (PMOs)have been demonstrated to effectively induce exon skipping and dystrophin expression in the Golden retrieval muscular dystrophic (GRMD) dog and the dystrophicmdx mouse. However, systemicdelivery of PMOs in both models also demonstratedthat expressionof dystrophin can not be achieved homogeneously in all muscle of the body withespeciallypoor inductionin the cardiacmuscleand diaphragm. Effortshave beenmadeto search for and achieveimproveddelivery and improvedantisenseoligonucleotideefficiency. In thisstudy, we examine the effect of PMOstagged at the 3 prime with an argininebased polymer (Vivoporter, supplied by GeneTools) on skipping of E23 of the dystrophin gene in the mdx mice in vivo. We found thatjust I0llg morpholino-vivoporter improvedlocalefficiencyof PMO induced exon skipping with almost 100%TA muscle fibres expressingdystrophin.Intraperitoneal injectionof the morpholinovivoporter achieved full induction of dystrophin in the diaphragm and abdomen muscles, but limited dystrophin expression in other skeletal and cardiac muscles. Since DMD severely affects respiratory muscles specifically the diaphragm, resulting in progressive deteriorations and failure of respiratory system, full induction of dystrophin in these musclesby i.p. injectionmay providea realistic alternative for the rescue of the respiratory functions in DMD.

Molecular Therapy Volu me 15.Supplement I. ~b y 2007 Co pyr ight © "1111: American S ociety o f Gen e Th erapy