340 The Anti Gene Oligonucleotide “Zorro LNA", Delivered by a Cationic Lipid, DownRegulates Huntingtin Gene Expression in Mammalian Cells Molecular Therapy Volume 20, Supplement 1, May 2012 Copyrigh[.]
OLIGONUCLEOTIDE & RNAI THERAPEUTICS 337 Effect of Argonaute-2 in Mediating NonCoding RNA Expression Paul N Valdmanis,1 Biswajoy Roy Chaudhuri,1 Dan Cao,1 Yannick Pouliot,1 Mark A Kay.1 Pediatrics, Stanford University, Stanford, CA The role of microRNAs is paramount for many cell processes This role is even more notable during embryo development when key spatio-temporal patterns of gene expression need to be maintained to potentiate proper tissue growth and positioning We have identified a set of microRNAs that are upregulated throughout mouse embryo development Interestingly, the most dramatic patterns of this increased expression occurs in imprinted domains and are dependent on the cleavage capacity of argonaute-2 (Ago2) to mediate their repression on certain target RNAs As an example of this, the reduction of expression of the Rtl1 gene is due to upregulation of microRNAs such as mmu-miR-127 that is transcribed in the antisense orientation To examine the change in gene expression we performed both small RNA (microRNA) high throughput sequencing and RNA sequencing on mouse embryo fibroblast cell lines This revealed a set of genes that are differentially expressed during development and that are influenced by microRNA expression and the availability of Ago2 Genes with predicted miRNA binding sites were cloned into luciferase vector systems and they were shown to downregulate 10 of 13 targets Further, we noted an accumulation of antisense transcripts in cells lacking functional Ago2 suggesting a role for Ago2 in using these antisense transcripts and cleaving and eliminating the sense counterparts Together, this indicates a network of transcriptional events that are established during development and maintained during certain adult tissues Understanding the requirements that utilize this RNA interference machinery is essential when evaluating the effect of oligonucleotide therapeutics and their potential for unintended effects on endogenous genes and microRNAs These results can be extended to cancer therapeutics where the role of antisense transcription has recently become better appreciated 338 Effective Regression of Melanoma Cancer by Delivery of Combinatorial siRNA Using an Arginine-Engrafted Biodegradable Polymer Jagadish Beloor,1 Chang Sun Choi,1 Boyong Choi,1 Minsun Park,1 Sung Hwa Kim,1 Andrew Jackson,2 Sungwan Kim,3 Priti Kumar,2 Sang-Kyung Lee.1 Bioengineering, Hanyang Universiy, Seoul, Korea; 2Internal Medicine, Yale University, New Haven, CT; 3Department of Pharmaceutics and Pharmaceutical Chemistry, University of Utah, Salt Lake City, UT Small interfering RNAs (siRNA) represent a relatively new class of molecules that are under clinical development for cancer therapy However, effective delivery of siRNA for the rapid and sustained regression of established tumors remains a daunting task Here, we investigate a combinatorial RNAi strategy for cancer therapy using an arginine-grafted bioreducible poly (disulfide amine) polymer (ABP) for the co-delivery of siRNAs targeting three anticancer genes Bcl-2, VGEF and Myc involved in discrete oncogenetic pathways Exposure to the ABP-complexed siRNA cocktail allowed extended control of B16.F10 melanoma cell proliferation in culture but also in vivo in a mouse model of solid tumor engraftment with the triple siRNA combination maximally inhibiting cell growth Both intratumoral and systemic administration rapidly regressed established large volume solid tissue tumors The preferential and enhanced accumulation of carrier-siRNA complexes in the tumor tissue enabled drastic reversion of tumor progression with individual siRNAs used at 0.1mg/Kg body weight and with just two administrations of the formulation Our results underscore the synergistic benefits of multiplexing inhibitory siRNAs in coalition with an efficacious systemic delivery platform for advanced stage solid tumor therapy Molecular Therapy Volume 20, Supplement 1, May 2012 Copyright © The American Society of Gene & Cell Therapy 339 Detection Methods for In Vitro and In Vivo Characterization of Cell-Specific, Cell-Internalizing RNA Aptamers for siRNA Delivery Luiza I Hernandez,1 William M Rockey,2 Craig A Howell,1 Xiu Ying Liu,1 James O McNamara II,1 Paloma H Giangrande.1,2,3 Department of Internal Medicine, University of Iowa, Iowa City, IA; 2Department of Radiation Oncology, University of Iowa, Iowa City, IA; 3Molecular & Cellular Biology Program, University of Iowa, Iowa City, IA Recent clinical trials of small interfering RNAs (siRNAs) have highlighted the need for robust delivery and detection techniques that will enable the application of these therapeutics to increasingly complex disease and organ systems RNA aptamers represent an emerging class of pharmaceuticals with great potential for targeted diagnostics and therapy More recently, these synthetic RNAs have been successfully used to deliver siRNAs to target cells in culture and in vivo While encouraging, the extended use of RNA aptamers as a delivery tool for siRNAs awaits the identification of RNA aptamer sequences capable of targeting and entering the cytoplasm of many different cell types Towards this end, we have developed novel cell-based selection approaches coupled to detection methodologies for identifying and characterizing cell-specific, internalizationcompetent aptamers for siRNA delivery Here we report on different methodologies, such as quantitative PCR and microscopy (fluorescence and confocal), for determining the rate of internalization and localization of these RNAs within the target cell Moreover, localization of a subset of these RNAs into the cytoplasm of target cells was confirmed using an RNA-RIP assay For this assay, the RNA aptamer is conjugated to a Ribosome Inactivating Protein (RIP) which, results in pronounced cell death upon inhibition of the ribosome complex within the cytoplasm of cells While assays performed on cells in culture are a necessary first step to identifying candidate internalizing RNA aptamers, the most critical step in the development of a potential drug is the determination of its disposition in vivo (e.g pharmacokinetics-PK/ pharmacodynamics-PD, toxicology) prior to administration to humans Here we describe linking chemistries for coupling RNA aptamers to infrared (IR) fluorescent dyes with optimal in vivo profiles Animals injected with the IR-labeled RNAs can be imaged while under anesthesia using a Xenogen IVIS 200 biological imager (or similar equipment), and the distribution of the IR-labeled RNA in the body can be visualized We demonstrated the use of this approach by determining the PK and biodistribution of the RNA aptamers in mouse models of cancer In conclusion, we describe novel methodologies for the rapid identification and characterization of RNA aptamers capable of delivering siRNAs into the cytoplasm of target cells We coupled these methodologies with state-of-the-art RNA chemistries and fluorescent technologies resulting in effective, targeted image-guided RNA reagents (TIGRs) that can be easily tracked in vivo Importantly, these TIGRs may represent a crucial first step in the transition of siRNAs from the bench-side into the clinic 340 The Anti-Gene Oligonucleotide “ZorroLNA”, Delivered by a Cationic Lipid, DownRegulates Huntingtin Gene Expression in Mammalian Cells Eman M Zaghloul,1 Pedro M D Moreno,1 Abdalla J Mohamed,1 Jesper Wengel,2 Karin E Lundin,1 C I Edvard Smith.1 Department of Laboratory Medicine, Karolinska Institute, Stockholm, Sweden; 2Department of Physics and Chemistry, University of Southern Denmark, Odense, Denmark Huntingtin (HTT) is a ubiquitous protein that has a crucial function in embryonic development and other biological processes Huntington disease (HD) is a severe neurodegenerative disorder caused by the increased number of CAG repeats leading to formation of a mutant S133 RNA VIRAL VECTORS HTT Aggregates of the mutant protein molecules seem to disturb the normal protein degradation pathways, and induce neurotoxicity in the brains of HD patients Existing therapeutics can improve the quality of life but they not significantly reverse or alter disease progression or increase life expectancy Studies in mice have recently shown that it can be beneficial to reduce the total HTT level to 20-30% in adult HD mice, even during treatment periods of up to months, which is a very long treatment period considering the life-span of a mouse These results were more recently confirmed in primates Here, we have utilized the newly developed anti-gene oligonucleotide (ON) “Zorro-LNA” to down-regulate the HTT expression in mammalian cells Zorro-LNA (Zorro) is a Z-shaped construct, originally made of LNA/DNA mixmers, with the ability of strand invasion and specific binding to its target sequence in duplex DNA (Figure 1) exceedingly complex, and therefore poorly understood Strong evidence suggests that an HIV-expressed long non-coding RNA antisense to the viral promoter plays a role in directing the epigenetic regulation of proviral gene expression and may thus contribute to the establishment and/or maintenance of viral latency We characterized the role of this HIV-1 specific long antisense RNA in modulating viral gene expression in CD4+ T cells as well as Ach2 and J1.1 cell lines Suppression of this transcript in latent viral human cell models and CD4+ T-cells resulted in increased viral expression that correlated with a loss of silent state epigenetic marks at the viral promoter This activation of viral expression was consistent with significantly higher viral p24 levels and correlated with a reduction in the antisense HIV-1 RNA Small single stranded RNAs designed to target the previously identified HIV-1 antisense promoter were also found to significantly activate HIV-1 expression in infected jurkat cells, suggesting that the U3 region of HIV-1 functions as an antisense promoter and is susceptible to small RNA-directed transcriptional gene silencing The data presented here suggest that the HIV-1 expressed antisense non-coding RNA epigenetically regulates viral expression via the endogenous cellular machinary required for RNA directed transcriptional gene silencing in human cells This non-coding RNA may be a key contributing factor in viral latency and a novel target for purging latent viral reservoirs in HIV-1 infected CD4+ T cells 342 New Approaches for Targeted Delivery of Dicer-Substrate siRNA We designed Zorro constructs, two of them targeting adjacent sequences in the 5’-untranslated region (5’UTR) and the other two targeting a stretch in the polyA1 region of the HTT gene Each pair of Zorros was transfected using a cationic lipid-based transfection reagent into Human Embryonic Kidney 293 cells Post transfection, RNA was prepared and multiplex RT-PCR was run to determine the remaining amount of HTT mRNA versus that of an endogenous control gene Interestingly, we have found that Zorros, in a final concentration of 50 nM, caused down-regulation of the HTT gene ranging from 40-60% Moreover, in time-course experiments, we have noticed that the onset of the blocking effect started already one day after transfection and lasted for four days (last time-point analyzed) Combination of Zorros targeting the two different sequences was markedly more efficient; 75% blocking of HTT was obtained Constructs utilizing the 2’-O-methyl RNA chemistry replacing the DNA bases as well as Zorros synthesized in the single-stranded form are currently being evaluated and compared to the original constructs Thus, the influence of different chemistries and synthesis strategies on the effect of HTT down-regulation will be also discussed In conclusion, we show for the first time, in this study, that Zorro-LNA delivered by a cationic lipid and via targeting DNA seems to downregulate the HTT gene expression in mammalian cells This finding represents a novel treatment strategy for HD 341 Activation of Latent HIV-1 by Targeted Suppression of an Endogenous HIV-1 Expressed Antisense Non-Coding RNA Sheena M Saayman,1 Kevin V Morris.1 Molecular and Experimental Medicine, The Scripps Research Institute, La Jolla, CA A major limiting factor of currently available anti-HIV therapies is their inability to eradicate latent pools of HIV-1 Despite longterm containment of viremia in patients receiving highly active antiretroviral therapy, latent reservoirs of virus persist in CD4+ T cells indefinitely before re-emerging The molecular mechanism underlying the establishment and maintenance of viral latency is multifaceted, S134 Kato Shum,1 Peixuan Guo,2 John J Rossi.1 Department of Molecular And Cellular Biology, Beckman Research Institute of City of Hope, Duarte, CA; 2Department of Pharmaceutical Sciences, University of Kentucky, Lexington, KY One of the key challenges facing the clinical translation of siRNA drugs is delivery, both systemic and to specific cells or tissue types A variety of small molecules, lipids, peptides and aptamers have been examined as potential delivery vehicles and vectors for siRNA Among these, we have previously developed dual inhibitory aptamerssiRNA conjugates that can selectively target B cell lymphomas and HIV-1 infected cells In this study, we describe a new approach for siRNA delivery using the non-viral trifurcate domain of bacteriophage phi29 RNA as platform for aptamers/siRNA delivery for the potential treatment of HIV-1 We make use of the trifurcate phi29 RNA system to serve as a nano-particle platform for the delivery of three therapeutic agents, 1.) a cell surface recognizing CD4 aptamer; 2.) a CCR5 dicer substrate siRNA and 3.) a tat/rev dicer substrate siRNA The CD4 binding aptamer will act as a targeting vehicle to facilitate selective intracellular access of the nanoparticles into HIV1 infected cells when CD4 is shuttled from the plasma membrane to the cytoplasm We describe the aptamer selection using proteinSELEX and cell-SELEX approaches to isolate 2’ fluoro modified RNA aptamers targeting CD4 from a combinatorial library with 40- nucleotide random sequence We also describe the assembly and characterization of dicer substrate siRNAs and aptamers using the phi 29 RNA nanoparticle platform We intend to extend this system to intramolecular aptamer-siRNA delivery as a proof-of-concept study RNA Viral Vectors 343 A Novel U3 LTR Attachment Site Deleted Non-Integrating Lentiviral Vector Aaron M Shaw,1 Troy B Hawkins,1 Kenneth G Cornetta.1 Medical and Molecular Genetics, Indiana University School of Medicine, Indianapolis, IN The risk of insertional mutagenesis continues to be an important issue when considering the risk: benefit when using lentiviral vectors (LVs) Non-integrating lentiviral vectors (NILVs) offer a potentially Molecular Therapy Volume 20, Supplement 1, May 2012 Copyright © The American Society of Gene & Cell Therapy ... time, in this study, that Zorro-LNA delivered by a cationic lipid and via targeting DNA seems to downregulate the HTT gene expression in mammalian cells This finding represents a novel treatment... cellular machinary required for RNA directed transcriptional gene silencing in human cells This non-coding RNA may be a key contributing factor in viral latency and a novel target for purging latent... 5’-untranslated region (5’UTR) and the other two targeting a stretch in the polyA1 region of the HTT gene Each pair of Zorros was transfected using a cationic lipid-based transfection reagent into