Abstracts 7 th Annual Meeting of the Oligonucleotide Therapeutics Society Copenhagen,Denmark September 8–10,2011

Over the last decade chemicaly modified nucleosides have recived a renewed focus with the development novel techniques in biochemistry such as RNAi. Especially UNA (unlocked nucleic acid) has been demonstrated to perform well in siRNA applications with lowered off-target effects and enhanced activity.^1,2 We herein introduce the 2′-amino-UNA derivative with an amino functionality at the 2′-position of UNA and present the synthesis of the 2′-amino-UNA phosphoramidite (9) from uridine (1) using the previously published selective benzoylation of the 2′-hydroxylgroup of UNA. ³

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FIGURE 1.

UNA, 2-Amino UNA and RNA.

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SCHEME 1.

Synthesis of 2′-Amino-UNA.

Functional sequestration of microRNA 122 (miR-122) by treatment with an oligonucleotide complementary to the microRNA results in long-lasting suppression of hepatitis C virus (HCV) viremia in primates. However, the safety of the constitutive miR-122 silencing approach to HCV inhibition is unclear, since miR-122 can modulate the expression of many host genes. Here, we try to develop a genetic regulation system capable of specifically inhibiting miR-122 activity only upon HCV infection. With an in vitro selection technique we developed an allosteric self-cleavable Hammerhead ribozyme, whose activity was specifically induced by HCV NS5B replicase such as to release the most efficacious antisense sequence to miR-122 only in the presence of HCV NS5B. The activity of the reporter construct with miR-122 target sequences at its 3′ untranslated region and the expression of endogenous miR-122 target proteins were specifically stimulated only in HCV replicon Huh-7 cells, but not in naïve Huh-7 cells, when transfected with the specific allosteric ribozyme, indicating that miR-122 activity can be specifically silenced by the allosteric ribozyme only in HCV replicating cells. Noticeably, HCV replicon replication was efficiently inhibited by the allosteric ribozyme. This ribozyme could be useful for the specific, safe, and efficacious anti-HCV therapeutic tool.

41 Are Short Oligonucleotides Less Specific In Vivo?

The challenge of developing effective and safe RNA-targeted therapeutics calls for robust in silico design of target-specific oligonucleotides with minimal effect on unintended targets. In this process, many molecular biologists and statisticians often ask how many perfect match recognition sequencesa given oligo-nucleotide has in the transcriptome. This, in turn, leads to the assumption that the longer a given oligonucleotide, the more specific it will be, whereas short oligonucleotides areoften considered unspecific and to have more off-target effects. However, this assumptionis only correct if the hybridization stringencycan be controlled in such a way that a single mismatch, an insertion, or a deletion will abrogate effective binding to unintendedtargets. This is indeed the case in most experiments such as PCR, Northern blot analysis, and in situ hybridization. The primary goal is to find experimental settings in which the oligonucleotidepreferably hybridizes to the intended target. In the lab, hybridization conditions such as temperature and ionic strength can be controlled to achieve optimal specificity. By comparison, hybridization conditionsin live animals or humans cannot be adjusted, instead altering the length, design and chemical modifications of a given oligonucleotide must be utilized to achieve adequate specificity.

Here, we present and compare different oligonucleotide specificity prediction algorithms. We show that different methods can produce qualitatively very different results. Algorithms based on minimizing mismatches predict that short oligonucleotides are less specific, whereas methods that maximize the number of basepairs or binding affinity to putative targets predict that longer oligonucleotides are less specific. We assess the aforementioned approaches using transcriptomal expression profiling of mice livers after treatment with different LNAphosphorothioate oligonucleotides.

The Argonaute proteins play important regulatory roles in the metabolism of cells and are involved in RNA interference (RNAi) mechanisms. In Caenorhabditis elegans and Arabidopsis thaliana, creation of Argonaute (Ago) mutant strains has allowed the study of their biological function, while in mammals this approach has been so far unsuccessful due to lethality of such mutations.

In human cells four different Ago paralogs are present (hAgo1-4) and the functional studies of these proteins relied on phenotypic suppression using small interfering RNA (siRNA). The knockdown is achieved by the activity of the Ago proteins themselves which are involved in the RNA interference mechanism. By suppressing the expression of hAgos using these tools, the obvious consequence is a dying out of the silencing effect, which could lead to interference between observed functional phenotypes and the mechanism of inhibition. Thus, alternative methods acting by different regulatory mechanisms would be advantageous in order to exclude unspecific effects. The knockdown may also be achieved by using specific antisense oligonucleotides (asONs) which act via an RNase H-dependent mechanism, not thought to interfere with the natural processes in which Agos are involved.

Functional observations in the use of hAgo-specific asONs indicate the relevance of this assumption and confirm that hAgo2 is involved in microRNA (miRNA) and in siRNA-mediated silencing pathways while the other hAgos play a role only in miRNA-based gene regulation (Mescalchin et al. RNA 2010 16:2529-2536).

Here, we describe the effects of hAgo-specific downregulation by asONs and compared them to those obtained by siRNA and shRNA. Moreover, we describe a novel strategy which allows functional studies of hAgos and it is based on the simultaneous downregulation of the endogenous proteins and overexpression of recombinant tagged and codon optimized hAgos.

50 Plasma Pharmacokinetics of Miravirsen in Humans

MiR-122, an abundant hepatic microRNA is an obligate host factor for HCV propagation and represents a unique target for Hepatitis C Virus (HCV) therapy. The pharmacokinetics of a LNA-modified antisense oligonucleotide complementary to miR-122, (miravirsen sodium), was studied after single or multiple doses (i.v.) or (s.c.) to healthy subjects. Subjects were divided into 6 groups (receiving 5 doses between 1.0 and 5.0 mg/kg/week). Each group consisted of 5 individuals (4 active and 1 placebo). Plasma concentration of miravirsen was quantitated using a validated hybridization-dependent ELISA. Plasma pharmacokinetics (e.g. AUC values) did not substantially change moving from a single dose to multiple doses and exposures after i.v. and s.c. administrations were similar. The plasma PK for miravirsen were multiphasic with distribution phase(s) and a terminal phase. The distribution phase was completed within 24 hours (t_½≈1-2 hours) and the plasma compartment was almost freed of miravirsen (to >99%). The terminal phase lasted for weeks (t_½=38 days). Comparison of AUCs following s.c. and i.v. doses indicated bioavailability was 100%. The terminal PK parameters (t_½, V_z and Cl) were similar independent of route. AUC_0-96h and C_max after s.c. administration increased dose –proportionally for AUC_0-96h, and less than dose-proportionally for C_max on both Days 1 and 29. The mean ratio of AUCday29/AUCday1 was 1.0, indicating that there was no accumulation after 5 doses. Terminal PK parameters were estimated from extended sampling (up to 140 days) after the last dose on Day 29. Total body clearance, Cl/F, was low and independent of dose, with a mean value of 36.0 (±8.5) mL/h/kg (all s.c. doses combined). The apparent volume of distribution, V_z/F, was large and independent of dose, with a mean value of 45.4 (±14) L/kg, indicating a distribution into a deeper compartment e.g. liver and kidney. Mean terminal plasma half-life was 38.3 (±16) days (all s.c. dose levels combined). While there was no significant accumulation of miravirsen in plasma, equilibrium or trough plasma concentrations increased linearly with dose and with number of doses, reflecting accumulation in deeper compartments. These data will be used to design dosing schedules for future clinical trials.

51 Towards Evaluation of Point-Mutations and Drug Interference with Mirna Regulated Expression of BRCA1

It is a challenge to try to understand how gene expression is regulated in various eukaryotic organisms, including the human body. The cells in eukaryotic organisms contain small double-stranded hairpin-like RNA structures coded for by microRNA (miRNA) genes in the cell nucleus. By use of the RNA induced silencing complex (RISC), the miRNAs interfere with translation by targeting mRNA. This kind of gene silencing can also arise if a small double-stranded exogenous RNA, so called small interfering RNA (siRNA), is introduced into a eukaryotic cell ¹ . The aim in the present project is to further increase the knowledge concerning the sensitivity of the siRNA and miRNA machinery towards interference caused by point-mutations and drugs. Strategies that are used involve in-house design of siRNAs and miRNAs and construction of drug-miRNA adducts for evaluation of drug interference. Potential drug candidates are used to investigate the expression of the BRCA1 gene, which codes for proteins that are associated with breast cancer development ² . In the initial studies, BRCA1 mRNA targeting has been evaluated in a luciferase-based assay (see Figure 1) and on protein level.

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FIGURE 1.

Measured firefly luciferase activity of MCF-7 lysates. Untreated MCF-7 cells were used as a control and the luminescence was normalized with the renilla luciferase signal. The treated MCF-7 cells were transfected (the protocol Cotransfecting plasmid DNA and RNAi into mammalian cells using Lipofectamine 2000 was used, Invitrogen) with 5 pmole siRNA (Qiagen, Hs_BRCA1_15 targeting the 3′-UTR of BRCA1), 5 pmole unplatinated has-miR-769-3p or 5 pmole platinated has-miR-769-3p per well (containing approximately 60 000 cells). Lysis was performed of the cells by using the active lysis method (Promega, Dual-luciferase reporter assay system) after the 39th incubation step.

Myostatin is a secreted growth factor that acts to negatively regulate muscle mass. Myostatin null mutations result in increased musculature in cattle, mice and humans and thus myostatin blockade is being considered as a putative therapy for muscle wasting disorders such as Duchenne muscular dystrophy (DMD). Various myostatin blockade strategies have been shown to induce functional improvement in the mdx mouse model of DMD. Similarly, the combination of myostatin blockade with dystrophin splice correction has results in greater functional improvement than either strategy alone. Transcriptional Gene Silencing (TGS) can be induced by small RNA molecules complementary to target gene promoter sequences. TGS effectors target chromatin modifying activities to low copy number transpromoter RNAs which induce silent state chromatin structure and, in some cases, promoter DNA methylation. Consequently, TGS may be permanent and heritable. In order to investigate the possibility of silencing myostatin by TGS we designed small interfering RNAs (siRNAs) targeting the myostatin promoter. These were transfected in differentiated C2C12 mouse myotubes. Two siRNAs resulted in significant myostatin silencing. Knock-down was observed in multiple transfection experiments, and with both conventional and nuclear-targeting transfection reagents. The results were replicated in the disease relevant H2K-mdx cell line indicating that the effect is not unique to C2C12 myoblasts. Myostatin silencing was also found to be independent of the interferon response. Treatment with the histone deacetylase inhibitor Trichostatin A alleviated the silencing effect of promoter-targeting siRNA suggesting that epigenetic remodelling at the myostatin promoter is responsible for the observed silencing. Similarly, enrichment of the silent state histone mark H3K9me2 was observed at the myostatin promoter following treatment with promoter-targeting siRNA.

We also report progress towards achieving myostatin transcriptional silencing in vivo.

53 A New Tool for Site-Specific Chemical Modification of RNA

The chemical modification of RNA has a strong impact on RNA research; the fluorescent labelling of RNA is useful to monitor RNA production, processing, relocation in the cell, interaction with other intracellular components and degradation, etc. Modified nucleotides in RNA play an important role in cellular functions and the progression of diseases. Thus, chemical tools that can modify nucleotides in RNA with high specificity and efficiency in situ may specifically alter the function of the target RNA, and thereby will be valuable for an RNA study as well as for manipulation of the gene expression at the RNA level.

We recently developed the efficient functionality-transfer reaction (FTR) for site-specific modification of RNA by using the ODN probe involving the 6-thioguanosine functionalized with the transfer group. ¹ RNA modification occurs at the 4-amino group of the cytosine at pH 7 or at the 2-amino group of the guanine base under alkaline conditions or in the presence of NiCl₂. The FTR was applied to pin-point labeling of RNA with a variety of functional groups such as biotin, fluorescent dyes and PEG, etc. through “click chemistry” (Fig. 1). ^1d Biotin-modification of RNA may have broad utility, because biotin can bring avidin-fused proteins to the specific site of RNA. In this paper, we discuss whether the functionality-transfer ODN can modify large RNA molecules such as mRNA, and what effects may be induced on the biological functions of the target RNA such as for polymerization and translation.

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FIGURE 1.

The functionality transfer reaction (FTR) for modification of the 2-amino group of guanosine of the internal RNA.

The innate immune response to viruses is based on pattern recognition receptors (PRR) that detect a characteristic structure or an unusual location of viral nucleic acids within the cell. As viral RNA is located in the same compartment as host RNA, PRRs in the cytosol have to discriminate between viral and self RNA by structure or modification. This challenging task is taken on by the cytosolic DExD/H-box family helicases RIG-I and MDA-5. The ubiquitously expressed receptor RIG-I detects 5’triphosphorylated RNA (pppRNA) occurring during viral RNA replication thereby controlling the immune response to most RNA viruses. Like viral RNA, eukaryotic mRNA harbours a triphosphate at its 5’end. So far it is presumed, that the N7-methylguanine (^m7G) cap structure, as present on all eukaryotic mRNAs, shields the 5’-triphosphate of self mRNA from RIG-I-mediated immune recognition. Here we synthesized RNA containing different cap structures and demonstrated that a ^m7G cap could only partially reduce RIG-I stimulation, while a G cap (without N7 methylation) did not impair RIG-I activation at all. By contrast, a single 2′O-methyl group at the 5’-penultimate residue (N1), a characteristic feature of vertebrate mRNA, was sufficient to abrogate RIG-I activation completely.

Interaction assays of recombinant RIG-I with modified 3P-RNA ligands confirmed that mainly the 2’O-methylation at N1 mediated inhibition of binding of capped RNA to RIG-I. Studies on the yellow fever virus-encoded 2′O-methyltransferase indicated that viruses mimic cap 2′O-methylation at N1 to escape RIG-I recognition. Thus, not the ^m7G cap but the 2′O-methyl group at N1 is the critical determinant for RIG-I to distinguish self from foreign triphosphate-containing RNA.

55 Antisense Approaches to Study REST/NRSF Transcriptional Regulation as a Therapeutic Target for Neurodegenerative Disorders

The repressor element-1 silencing transcription factor/neuron-restrictive silencer factor (REST/NRSF) is a master regulator of neuronal gene expression. REST functions through binding the genomic loci that contain the repressor element-1 (RE1) binding motif and recruits a series of epigenetic and regulatory cofactors to its N- and C-terminal domains. Various studies have characterized the specific roles played by REST during neuronal lineage specification and maturation. Among the REST targets BDNF has emerged as a candidate of interest as a potential neuroprotective and functionally restorative treatment in psychiatric and neurological disorders, including Huntington's disease (HD). In HD, BDNF levels are significantly attenuated as a result of impaired BDNF transport from the cortex to the striatum in the presence of mutant HTT. Wild-type HTT also regulates transcription of BDNF through REST, by sequestering REST in the cytoplasm, therefore restricting its access to the nucleus, which leads to the transcription of target genes such as BDNF.

In this study, we were interested in down-regulation of REST as a potential therapeutic strategy to both slow down cell death and maintain the functional state of remaining neurons. This could be possible through the neuroprotective and functional effects of BDNF by activation of signaling in neuronal pro-survival pathways. In order to specifically target REST, we applied 2^nd generation Antisense Oligonucleotides (ASOs) to reduce levels of REST mRNA in the liver and the CNS in both normal (wild type) and HD mice and characterized the effects on BDNF expression and gene expression more globally, as determined by microarray analysis.

We have demonstrated specific reduction in REST levels in both liver and CNS following treatment by ASOs, which resulted in the induction of a number of neuronal genes including BDNF and Synapsin1 at the mRNA and protein levels. Gene array expression analysis was performed on BALB/c mouse liver and R6/2 brain, a mouse model of HD, following systemic and ICV administration of REST ASO, respectively. Samples were analyzed by hybridization to the MouseWG-6 v2 Expression BeadChip array (Illumina), to gain insight into putative pathways affected by REST suppression. Array data analysis was performed using GeneSpring, followed by Gene network prediction using Ingenuity™ Pathway Analysis (IPA). 416 genes were found to be up regulated and 119 genes down regulated following REST ASO treatment. Numerous novel genes were identified that were affected by REST suppression that are predicted to play a role in cancer, genetic disorders, neurological diseases, cell-to-cell signaling, and tissue development.

Our findings suggest that REST may be an important target for neurodegenerative diseases like HD, that it is also involved in the regulation of a broad range of cellular pathways outside of the CNS, and that the antisense approach is a viable strategy for selectively modulating REST activity in both the CNS and in the periphery.

56 Functional Analysis of Micrornas as Regulators of Cargo Trafficking

MicroRNAs (miRNAs) are small non-coding RNAs emerged as important translational gene expression regulators in eukaryotic cells. miRNAs are predicted to modulate the expression of nearly 60% of protein-coding mammalian genes. Thereby, miRNAs affect a multitude of biological processes such as developmental transition, cell proliferation and apoptosis. Alteration of miRNA expression patterns leads to tumorigenesis, diabetes, neurodevelopmental disorders and many other diseases.

In the present study we demonstrate an unbiased integrative approach to identify miRNAs and their functionally relevant target genes involved in the regulation of secretory membrane trafficking. By applying (i) molecular biology, (ii) fluorescence microscopy, (iii) statistical data analysis, (iv) bioinformatics and (v) miRNA and mRNA expression profiling methods we are enabled to identify miRNAs of interest. A high-throughput screening of oligonucleotides mimicking 470 human miRNAs led to the identification of 44 miRNAs with an effect on the model cargo protein ts-O45-G secretion rate. In parallel, a large-scale library screening of 875 miRNA inhibitors yielded 68 miRNAs, which inhibition also caused significant changes of cargo trafficking. Further examination of primary miRNA hits by combined microscopy-based and automated image analysis-based Golgi complex integrity assay revealed that perturbation of 13 miRNAs resulted in quantitative changes of Golgi complex morphology. Moreover, we uncovered an active role of oncogenic miR-17∼92 cluster in the regulation of cargo trafficking and identified TBC1D2 and LDLR genes as novel targets of miR-17.

In perspective, the results of detailed analysis of identified miRNAs and their target genes will allow us to model the posttranscriptional miRNA-mediated regulatory networks. A comprehensive understanding of how these networks coordinate the activity of secretory membrane trafficking as a global adaptive response system is needed to study miRNAs as promising therapeutic agents and targets under particular pathological conditions.

57 Silencing the CDK4 and CDK6 Genes Through RNA Interference

Alzheimer's disease (AD), a disorder which affects millions people worldwide, is characterized by progressive memory loss and a spectrum of behavioral abnormalities leading to dementia. An effective treatment of AD is still not available. Preventing or slowing down progression of neurodegeneration will considerably reduce the number of severely affected patients and improve the quality of life of the aging population. The biological causes of neuronal death in AD have been the objective of intense studies and several concepts were proposed [1]. Several investigators observed close association between the neuronal cell death and reactivation of the cell cycle in post-mitotic neurons. Abnormal cell-cycle-related proteins expression, as cyclin dependent kinases, cyclins and cdk inhibitors was observed in pathologically affected parts of AD brains [2]. Usually, in postmitotic and terminally differentiated neurons, the cell-cycle activity is arrested by the enrichment of cdk inhibitors, neurons are “locked” in the G0 phase. At the beginning of neuropathogenesis, neurons leave the G0 phase and progress until the S phase and die at the G1-M transition. The aim of our studies was to down-regulate the expression of two cyclin dependent kinases CDK4 and CDK6 in neural cells through RNA interference-based gene silencing. Such approach could be the proposal for the new neuroprotective gene therapy in AD and the other neurodegenerative disorders. Thus, sixteen siRNAs with sequences complementary to human, mouse and rat transcripts of CDK4/6 genes were designed and synthesized in house. Their silencing activity was screened in human (HeLa, SH-SY5Y), mouse (Neuro2a, RAW 264.7) and rat (PC12) cell lines and assessed by quantitative RT-PCR (target mRNA level) and by Western blot (target proteins level) analyses. Some of the silencers dowregulated the expression of both target genes up to 90%. Activity of siRNA was also valuated by flow cytometry, where we counted the number of cells in G1/G0, S and G2/M phases. Analysis has shown that for cells transfected with active siRNA molecules, which result in down-regulation of CDK4/6 genes expression the number of cells in G1/G0 phaseis is increased, while the number of cells in S phase is lowered. This effect dependented on concentration of siRNA. Selected sequences of the most active siRNAs were used for construction of shRNA and micro RNA based shRNA inserts cloned into two type of plasmids: with U6 promoter (Pol III) (pSilencer-2.0 U6, Ambion) and CMV promoter (Pol II) (pEGFP-C1, Clontech and pcDNA 6.2-GW/miR, Invitrogen). For selected cells transfected with these constructs the lowering of CDK4 and CDK6 genes expression was observed in the range 10-70% (mRNA) and 20-70% (protein).

59 Dicer-Substrate siRNA Exhibit Improved Guide Strand Selection and Stronger RISC Loading Complex Formation Compared to Canonical siRNA

As small interfering RNA (siRNA) continues making headway toward therapeutic applications, researchers are looking to optimize the efficacy of both a suitable delivery vehicle and the therapeutic RNA itself. We are interested in maximizing the efficacy of siRNAs at the RNA level through relatively minor chemical and structural modifications to canonical siRNA. The rationale behind modifying the canonical siRNA structure is, among other criteria, to improve loading of the guide strand into the RNAi machinery, reduce off-target effects, and possibly reduce competition with endogenous miRNAs, ultimately leading to better efficacy. Here, we present a parallel analysis of four siRNA variants (the canonical 21mer, a commonly used 19merTT, a Dicer substrate interfering RNA (dsiRNA), and a short trigger 16merTT)) in gene silencing, RNAi protein binding, and miRNA competition disruption assays. The dsiRNA consistently demonstrated better gene silencing by the guide strand and worse gene silencing of the passenger strand compared to its analogous 21mer or 19merTT. The dsiRNA also showed more robust formation of a high molecular weight complex known to contain Dicer and TRBP (two primary members of the RISC-loading complex) compared to its analogous 21mer and 19merTT. Small RNA deep sequencing results reveal that (1) cells transfected with dsiRNA have a global miRNA profile more similar to untransfected cells compared to 21mer- or 19merTT transfected cells, and (2) at low transfection concentrations, the guide strand of the dsiRNA is more abundant than the 21mer or 19merTT. The 16merTT did not perform comparably in any of the assays. Also, we are interested in re-exploring the 5’ triphosphate group on siRNAs that would trigger RIG-I-mediated interferon production as a bifunctional way to ablate diseased cells. Interestingly, preliminary results using 5’ triphosphate 19+2 siRNAs generated by T7 in vitro transcription did not silence reporter gene expression; we are exploring this further. While delivery of therapeutic small RNAs is arguably the largest hurdle to overcome, our studies show that relatively small changes to canonical siRNAs can also improve potency.

61 Kinetic Comparison of Gene Silencing Profile of siRNA and Shrna-Expressing Plasmid DNA In Vivo

RNA interference (RNAi) is a gene silencing event in which small interfering RNA (siRNA) degrades target mRNA in a sequence-specific manner. RNAi can be also induced by introducing plasmid DNA (pDNA) that expresses short-hairpin RNA (shRNA) into cells. Because of the specificity and potency of RNAi-mediated gene silencing, the research for the development of RNAi-based therapy has been an exciting field since the discovery of RNAi. For therapeutic application of RNAi, the time course of gene silencing is one of the important factors to be considered. However, little attention has been paid to the duration of RNAi-based gene silencing in vivo. There has been no report of gene silencing 1 week or longer after the induction of in vivo RNAi. In the present study, we evaluated and compared the profiles of gene silencing induced by siRNA and shRNA-expressing pDNA in mice. A newly developed reporter pDNA (pROSA-gLuc) encoding Gaussia luciferase cDNA under the control of human ROSA26 promoter was used to obtain a prolonged expression of the reporter protein over three months. We administered this pDNA to mice together with siRNA or shRNA-expressing pDNA targeting Gaussia luciferase at varying doses by the hydrodynamic injection method. The serum level of Gaussia luciferase was greatly but transiently reduced by siRNA, whereas a sustained reduction was obtained with shRNA-expressing pDNA. A quantitative analysis of the gene silencing profiles based on a moment analysis confirmed the finding that shRNA-expressing pDNA suppresses the target gene expression longer than siRNA. These results provide important information about the time-courses of RNAi in vivo.

63 Targeting Tumor Antigens to Dendritic Cells In Vivo Using Receptor-Specific Aptamers Conjugated to Tumor Antigen

The success of immunotherapy depends on the induction of effective primary immune responses to treat existing cancers and the generation of subsequent memory responses to prevent recurrence at a new site. Dendritic cells (DC), recognized as major antigen presenting cells, are uniquely equipped to initiate and regulate immune responses, making them a key target for developing new therapies. Immunizing with antigen-loaded DC is a powerful method of inducing CD4+ and CD8+ T cell responses and antibodies (Abs). Although ex vivo DC therapy has shown promise, it is a customized, complex, patient-specific cell therapy that reduces its universal applicability for cancer. Thus there is a critical need to develop strategies that circumvent the need for ex vivo DC loading. Directly targeting antigens to DC in vivo will facilitate the development of a more effective “off-the-shelf” tumor vaccine, which will be accessible to large numbers of patients.

DC express a number of specialized endocytic receptors of the C-type lectin family enabling capture of antigen by receptor-mediated endocytosis at very low concentrations. Studies exploiting the endocytic properties of DC receptors demonstrated that antigens conjugated to receptor targeting moieties, such as the natural receptor-ligands or receptor-specific antibodies, increased antigen capture and presentation 100-1000-fold as compared to soluble antigen. Moreover, receptor targeting facilitates cross-presentation to induce Ab and CD4+ T cell immunity as well as elicit MHC-class-I-restricted CD8+ or cytotoxic T lymphocytes (CTL), critical for cancer immunotherapy.

To deliver antigens to DC in vivo, we developed artificial receptor ligands to the macrophage mannose receptor (MMR or CD206), an endocytic C-type lectin receptor expressed on DC and macrophages. Herein, a RNA aptamer library comprised of 40 randomized nucleotides and modified with 2′Fluoro-modified pyrimidines for improved nuclease resistance was selected for affinity binding to MMR. A complex selection scheme with alternating rounds on both, recombinant human and murine MMR, as well as on cells engineered to express human MMR enabled us to identify cross-reactive aptamers that recognize MMR with high affinity. A truncated, minimal version of the identified lead-aptamer with retained receptor-binding properties gets rapidly internalized into human monocyte-derived DC in vitro. Currently we are testing aptamer-tumor antigen chimeras and aptamer-decorated nanoparticles to deliver model tumor antigens to DC in vitro and in vivo and investigate their capability to elicit an antigen-specific immune response.

RIG-I has been shown to be involved in sensing every members of the flavivirus genus. Yellow Fever Virus (YFV) possesses mechanism to overcome innate immunity. Here we propose the potent antiviral activity of RIG-I ligand (3pRNA). We used a sub genomic Yellow Fever Renilla Replicon (YFRR) model. Our Luciferase data showed that, 3pRNA inhibits one log replication of YFRR in HepG2 cells at non-toxic concentration (800ng/ml). MTT assay ruled out the detrimental effect of 3pRNA on HepG2 cells and the inhibition of replication is 3pRNA specific in dose dependent manner. We served poly A RNA in HepG2 cells as negative control and we did not observe inhibition of replication that, supports the antiviral activity of 3pRNA. 3pRNA additionally induces type 1 IFN in addition to the YFRR itself in HepG2 cells that leads to the inhibition of replication. Type 1 IFN and IP-10 were highly expressed in 3pRNA treated cells. 3pRNA did not show inhibition of replication in IFN deficient Vero cells. However, addition of rec-IFNβ showed inhibition of YFRR in Vero cells that suggests that, antiviral effect of 3pRNA is due to enhancement of IFN production and IFN signaling pathway mediated ISGs activation. WB and IF data showed that 3pRNA upregulated RIG-I and IRF3 and strongly inhibited NS4B protein of YFRR. Our real time-PCR data showed that 3pRNA potentially upregulated IFN dependent antiviral genes (OAS, RNAse L, MxA, IFIT1 but not IFIT2). Small form of PARP13 (ZAPs) is highly upregulated by 3pRNA which sparks the RIG-I signaling. 3pRNA did not inhibit replication in RIG-I-/- MEFs that clearly indicates that, inhibition of replication is RIG-I ligand dependent. RIG-I-/- MEFs showed enhanced replication compared to WT-MEFs. Over expression of Flag-tagged RIG-I showed non significant inhibitory effect on replication that also support our idea that 3pRNA is potent immune stimulator. Plaque assay showed that, 3pRNA inhibited about 65% virus particles of 17-D in infected HepG2 cells. Equivalent unit of rec-IFNβ with 3pRNA induced IFN-β showed almost similar negative effect on replication.

Altogether, these findings raise the possibility that RIG-I ligand, a potent immune stimulator may be a promising antiviral therapeutic RNA molecule for YFV and other flavivirus.

69 Glucan Particles for Selective Delivery of siRNA to Phagocytic Cells in Mice

Phagocytic macrophages and dendritic cells are desirable targets for potential RNAi therapeutics because they often mediate pathogenic inflammation and autoimmune responses. We recently engineered a complex, 5 component glucan-based encapsulation system for siRNA delivery to phagocytes. In experiments designed to simplify this original formulation, we discovered that the amphipathic peptide Endo-Porter forms stable nanocomplexes with siRNA that can mediate potent gene silencing in multiple cell types. In order to restrict such gene silencing to phagocytes, a method was developed to entrap siRNA/Endo-Porter complexes in glucan shells of 2-4 microns diameter in the absence of other components. The resulting glucan particles containing fluorescently labeled siRNA were readily internalized by macrophages, but not other cell types, and released the labeled siRNA into the macrophage cytoplasm. Intra-peritoneal administration of such glucan particles containing siRNA/Endo-Porter complexes to mice caused gene silencing specifically in macrophages that internalized the particles. These data indicate that specific targeting to phagocytes is mediated by the glucan, while Endo-Porter peptide serves both to anchor siRNA within glucan particles and to catalyze escape of siRNA from phagosomes. Thus we have developed a simplified siRNA delivery system that effectively and specifically targets phagocytes in culture or in intact mice.

70 Development of an RNA Aptamer for Human Toll-Like Receptor 2 Ectodomain

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, recognize pathogen-associated molecular patterns and play a crucial role in innate immunity by mediating the activation of macrophages. They also recruit phagocytic cells and produce proinflammatory cytokine/chemokine. Recently, it was suggested that activation of TLRs contributes to the development of certain cardiovascular diseases like atherosclerosis, congestive heart failure and cardiac dysfunction in sepsis. The cDNA encoding TLR2 was cloned from U937 cells using RT-PCR procedure. Human TLR2 (hTLR2) has 23 leucine rich repeats (LRRs) including a C-terminal LRR and two N-terminal LRRs. LRR9-LRR12 domain binds to ligands, whereas LRR11-LRR14 domain make contacts with hTLR1. To develop an RNA aptamer for hTLR2, LRR8-LRR15 domain was chosen as target protein and its coding sequences were cloned into pQE-80L vector that can express membrane protein with accompanying 6 his-tag sequences. Origami strain of E.coli that can efficiently express membrane protein was used for overexpression of LRR 8-15 domain protein. The purified protein was then used for in vitro selection of RNA aptamer(s) from the RNA library using SELEX procedure. Characterizations of the obtained aptamers are currently underway and preliminary results including electrophoretic mobility shift assay and minimization data will be presented at the meeting.

71 Arrest of the Elongation of Translation by Peptide Nucleic Acids

Oligonucleotides targeting 5’UTR of mRNA are often used to inhibit translation initiation. In cell-free system, Peptide Nucleic Acids (PNAs) targeting the coding region and forming triplexes are able to stop the ribosomal machinery during the translation elongation. We have shown that it is possible to successfully target mixed sequences in structured regions of messenger RNA with short PNAs that form duplex and triplex structures that arrest elongating ribosomes and induce truncated proteins synthesis^{1, 2, 3}. PNA is unique among the investigated steric blocking agents in its ability to specifically inhibit the translation elongation. In cells, dominant negatives could be thus generated thanks to PNAs.

Type I insulin-like growth factor receptor (IGF-IR) has been implicated in the progression of a large number of cancers and represents an important target for anti-cancer strategies. Studies showed that transfected dominant negatives of IGF-IR are able to decrease tumor growth and metastasis formation. We have shown that a PNA sequence forming a partial triplex on the coding region of IGFIR's mRNA induces a truncated IGF-IR in cell free system and inhibits the receptor expression in cultured prostate cancer cells. Therefore, we have shown that a steric blocker can stop the translation elongation in cells. In addition, IGF-1R knockdown by PNA led to an attenuation of phosphorylation of downstream signaling pathways, PI3K/AKT and MAPK, involved in survival and mitogenesis and also to a decrease in cell transformation ⁴ . Of the steric blockers tested, which included phosphorodiamidate morpholino oligomers and locked nucleic acids, PNAs was unique in its ability to form triplex structures with mRNA and to arrest translation elongation.

72 RNAi-Based Pancreatic Cancer Therapy by Local Delivery of Anti K-RAS^MT siRNA

One of the major challenges for oligonucleotides based therapeutics is delivery. Obstacles in systemic delivery include targeting, over-dosing and dose fluctuations, liver aggregation and immune-stimulation of the drug carriers, renal clearance and cost. Direct injection doesn't solve such issues in many cases. Local and prolonged delivery overcomes these hurdles and offer an attractive solution at least for a sub group of regional diseases, amongst are solid tumors. Pancreatic cancer is an aggressive disease, being the 4^th leading cause of cancer–related death in the western world. Genetic alterations in KRAS signaling pathway are involved in 95% of cases, while the majority of KRAS mutations are gain-of-function mutations at codon 12 (KRASG12D) of the oncogene. The tumor is addicted to KRAS activity. We developed an implanted controlled drug delivery system called LODER® (Local Drug EltueR), a miniature biodegradable polymeric matrix encompassing anti KRASG12D siRNA drug and preserving it against degradation. . The LODER® is implanted by endoscopic ultrasound within the tumor and releases drug regionally, along 8-10 weeks. Treatment of pancreatic cell cultures with siG12D LODER resulted in a significant inhibition of KRAS expression both on mRNA and protein levels. This inhibition of expression was associated with a decrease in cell proliferation and induction of cell death. In vivo the growth of human pancreatic cancer cell lines (PANC1 and CAPAN1) was retarded, both in subcutaneous and orthotopic models. Furthermore, the survival of mice implanted with siG12D LODERs was significantly improved in the two cell lines tested in the orthotopic mouse model. Based on these results, Silenseed has initiated a phase I study with siG12D LODER implanted into patients with a locally advanced pancreatic tumor: http://clinicaltrials.gov/ct2/results?term=silenseed.

Chronically activated pathogenic effector memory type 1 T helper (Th1) cells participate in immune-mediated diseases such as autoimmunity or chronic inflammation. These cells are resistant against physiological regulation and conventional immunosuppressive therapy by probably reducing the expression of the pro-apoptotic proteins BIM and Pten. To identify microRNAs (miRNAs) which regulate the expression of BIM and Pten and therefore regulate the persistence of pathogenic Th1 cells we compared the miRNA expression profiles of either acutely (once) or chronically (repeatedly) activated memory Th1 cells. We identified a candidate miRNA being induced in pathogenic effector Th1 cells. Computational target analysis via TargetScan and PicTar predicted BIM and Pten as putative targets. Overexpression of this miRNA in activated Th1 cells results in 50 % down-regulation of endogenous Bim and Pten expression. Conversely, inhibition of the candidate miRNA in chronically activated memory Th1 cells by antagomirs results in induced protein levels of BIM and significantly enhanced cell death and reduced proliferation. With its targets having important roles for survival and proliferation of pathogenic effector memory Th1 cells, this candidate miRNA represents a promising molecular target for the treatment of immune mediated disease.

75 Identification of Process Related Impurities in DNA/RNA by LCMS Analysis

LCMS is a powerful tool for identification of impurities in DNA/RNA based API's. Replacing ammonia with methyl amine in the deprotection process of a DNA thiolate gave rise to a new set of impurities. LCMS analysis showed that these impurities were formed by transamination of cytidine by methyl amine. The progress of methyl amine deprotection of acetyl protected dC/rC containing sequences was followed by LCMS. The data presented here show that the transaminated impurities arise from the deprotected cytidine and not the N-acetyl protected species and that this reaction is general to other alkyl amine based deprotections.

76 Design of Protein-Responsive shRNA System for Translational Control in Human Cell

We have developed an artificial protein-responsive shRNA switch by employing short hairpin RNA (shRNA) that serves as a powerful tool for RNAi in human cell. This artificially designed molecular systems for responding to particular gene products and rewire gene expression networks in living cells are highly useful.

As a pilot system, we have developed a synthetic shRNA (Kt-shRNA) that combine target sequences (double strand region) for target mRNA with Box C/D kink-turn RNA motif set in its loop region. An Archaeal ribosomal protein L7Ae and its binding motif, Box C/D kink-turn RNA motif, were employed as the RNP interaction motif for protecting Kt-shRNA from Dicer cleavage. Kt-shRNA responds to cellularly expressed L7Ae and controls target gene expression to promote apoptosis of human cancer cells. (Saito H. et al, Nat. commun., 2 : 160 (2011) ).

To develop new protein-responsive shRNA switches, we employed 3D molecular modeling software for adequately positioning the protein-binding motif in order to properly control Dicer activity. We were able to build new protein responsive shRNA switches with RNP motifs consisting of U1A or NFκB for the control of target gene expression. Thus, we succeeded in improving the ability of new shRNA switches by tuning their RNP interaction phases in silico. In conclusion, the protein-responsive-shRNA switch could serve as a useful tool for RNAi therapy or regenerative medical techniques, because a desired RNA motif binding to a disease or differentiation marker protein could be incorporated into the loop region of the synthetic shRNA for the control of a target mRNA translation.

78 Integrated Microfluidic Platform for Multiplex Selex on a Chip

Multi-target aptamer selection called Multiplex SELEX (Systematic Evolution of Ligands by EXponential enrichment) gains its interests rapidly because of its potential to high-speed and high-throughput aptamer selection capacity. Parallelization of chemical process by integration of microfluidic components can be a key technique to develop a multiplex SELEX process. One of the potential problems in multplexing chemical processes on-chip is cross-contamination. In order to avoid this consideration, we propose a microfluidic network platform with a pneumatic valving strategy to allow serial loading and incubation of aptamers with sol-gel entrapped target proteins. After target binding inside the sol-gels cross-contamination free parallel elution of specifically bound aptamers is performed. The platform allows selective binding against five different targets immobilized in sol-gel spots. When eluting bound species liquid flow across nearby reaction chamber are sealed by integrated pneumatically driven rubber microvalves. We demonstrate specific aptamer binding to its respective protein target and subsequent aptamer elution without any cross contamination. The current proof of concept opens the route to increased automation and microscale parallel processing of the SELEX methodology.

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FIGURE.

Concept of parallel process of aptamer binding and partitioning. Five different targets included sol-gel were spotted on the surface (Heater electrodes). Then, RNA mixture (Aptamers) were injected and incubated by fluidic operation for binding aptamers to their target proteins. Bound aptamers were collected by applying heat. (Pneumatic valves are operated during heat process in order to remove cross contamination).

Specific gene silencing using RNA interference (RNAi) has proven to be a useful tool in the fields of biotechnology and a promising future therapeutic modality. Small interfering RNAs (siRNAs) are used as a molecular trigger of RNAi to specifically block the expression of the genes causing various diseases including cancer. In this study, we chose KRAS (ki-ras2 Kirsten rat sarcoma viral oncogene homolog) as a target gene to develop anticancer siRNA therapeutics. KRAS belongs to a group of small GTP-binding proteins, and involved in cell signaling and growth. Therefore, by targeting KRAS through the RNAi, we expect repression of tumor survival and proliferation. The conventional siRNA structure shows several limitations such as off-targeting effects and inefficient gene silencing for certain genes. To overcome this, we developed various long antisense siRNA structural variants which harbor long antisense 3’ overhang sequence complementary to KRAS mRNA. We show that long antisense siRNAs can trigger improved gene silencing over conventional siRNAs. Long antisense structure also improved gene silencing triggered by asymmetric shorter-duplex siRNAs (asiRNAs). Our results demonstrate that long antisense siRNA can be an alternative structure to conventional siRNA with improved efficacy and specificity in gene silencing.

80 Stealth Delivery of Oligonucleotides

Nucleic acid delivery into live cells has been one of the major hurdles for oligonucleotide-based therapeutics. Although various approaches and methods have been developed and are currently being utilized, a new platform, Xyton Stealth Delivery^TM (XSD), has been developed for intracellular delivery of oligonucleotides with the aim of expanding therapeutic applications. Unique attributes of this new methodology include the formation of molecularly dispersed solutions and nearly maximal cellular uptake in the presence of serum within 90 minutes by a variety of cell types including primary hematopoetic cells. Furthermore, cellular entry of both single strands and duplexes of either DNA or RNA by this mode of delivery is nontoxic, homogeneous, and sequence-independent. When XSD was applied to transport of 24-mer antisense oligonucleotides, intracellular hybridization with complimentary sequences was shown to be saturable. Furthermore, Dicer substrates designed by the XSD technology were processed by the recombinant human enzyme; upon addition of a Dicer substrate targeting the eGFP gene to primary murine transgenic eGFP PBMC in serum, the expected siRNA effects were observed after 3 days as judged by flow cytometry. In summary, XSD delivery into the cytosol of both antisense DNA and RNA duplexes was uniform and rapid in a variety of target cells with mRNA hybridization and Dicer processing followed by specific gene knockdown, respectively. The delivery method described herein utilizes a totally unique class of delivery vehicles relative to what has been utilized in the RNAi and microRNA communities to date.

An oligonucleotide (ON) with a sequence complementary to a specific mRNA can inhibit its expression. The use of unmodified ON for applications in cells, however, is very limited because of the poor cellular uptake efficiency, targeted delivery, its low specificity and affinity for the target sequence, and susceptibility to degradation by nucleases. Many modified ONs containing unnatural bases or modified sugars have been prepared for improving the efficacy of the antisense method. One such example is ONs incorporating a 2′-modified nucleotide. These analogues are known to exhibit high binding affinity to target RNA and metabolic stability. Furthermore, cross-linking reactions are expected to enhance the antisense activity by irreversibly binding to the target RNA, based on the steric blocking mechanism. We previously demonstrated that the 2′-O-methyl (2′-OMe) analogue of 2-amino-6-vinylpurine (2-AVP) (1) reacted selectively to thymine in DNA, but did not react effectively to uracil in RNA under neutral conditions. ¹ In this study, we investigated about the effect of adjacent bases with 2-AVP on the cross-linking reactivity. In consequence, we found that ON incorporating (1) reacted more effectively to uracil under neutral conditions in the cases where the ON had the sequences 3’ G (1) C5’. Herein, we describe the neighboring bases effect on the cross-linking reactivity of 2′-OMe analog of 2-AVP, and evaluation of the antisense effect with the use of 1 in non-cell translation assay and in cell experiments.

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FIGURE 1.

Design of the 2-Amino-6-vinylpurine (2-AVP) (1) and Dependency of the Reactivity on the Neighboring Bases of (1).

Interleukin-17 (IL-17) is a pro-inflammatory cytokine produced primarily by a subset of CD4⁺ T cells, called Th17 cells, that is involved in host defense, inflammation and autoimmune disorders. Two IL-17 family members, IL-17A and IL-17F, are most structurally related, and form homodimeric (IL-17A/A, IL-17F/F) and heterodimeric (IL-17A/F) structures. We have isolated RNA aptamers against human IL-17A. Aptamers are short single-stranded nucleic acid sequences that are selected in vitro based on their high affinity to a target molecule. One such aptamer, Apt21-2, blocked the interaction between IL-17A and its receptor IL-17R in vitro, and inhibited the production of interleukin-6 from mouse and human cells. A PEGylated form of Apt21-2 inhibited the development of GPI-induced rheumatoid arthritis and MOG-induced experimental autoimmune encephalomyelitis in a dose-dependent manner, showing its therapeutic potential in autoimmune diseases.

Among several dimeric conformers of IL-17, Apt21-2 recognizes both IL-17A/A and IL-17A/F as does the known anti-IL-17A antibody. Although the biological significance of IL-17A and IL-17F have been investigated using respective antibodies or gene knockout mice, the functional study of IL-17A/F heterodimeric form has been hampered by the lack of an inhibitory tool specific to IL-17A/F. Hence, we aimed to develop an RNA aptamer that specifically inhibits IL-17A/F. One such aptamer against human IL-17A/F, AptAF42, was isolated through the subtractive SELEX procedure employing counter-selection against IL-17A/A. AptAF42 and its optimized derivative AptAF42dope1 blocked the binding of IL-17 receptor to IL-17A/F, but not to IL-17A/A or IL-17F/F, in the surface plasmon resonance assay in vitro. Consistently, AptAF42dope1 did not affect cytokine GRO- α production induced by IL-17A/A or IL-17F/F, but blocked IL-17A/F-induced GRO- α production in human cells. RNA footprinting assay of AptAF42dope1 using ribonucleases in the presence or absence of IL-17A/F revealed that part of the predicted secondary structure fluctuates between alternate forms and that AptAF42dope1is globally protected from ribonuclease cleavage by IL-17A/F. These results suggest that the selected aptamer recognizes a global conformation specified by the heterodimeric surface of IL-17A/F.

84 A Novel Assay for the Detection of miRNA Maturation

During the past years interest has increased in finding ways to manipulate miRNA function, not only in order to elucidate its mechanisms, but also with the perspective of affecting human disease formation and progression.^[1] In order to investigate the activity of potential inhibitors and modulators of Dicer-mediated miRNA maturation, a facile and accurate assay is needed.^[2,3] Here we provide a novel rapid and reliable assay of Dicer-mediated miRNA maturation, without the need of tedious and expensive labeling of pre-miRNAs. The assay is based on the rolling circle amplification (RCA) method,^[4] wherein only the mature miRNA but not the pre-miRNA can initiate isothermal amplification of a cyclic DNA template leading to the formation of a linear single stranded DNA (ssDNA) product. A secondary primer binds the product sequence and initiates the formation of a secondary DNA strand resulting in an enhanced fluorescence signal of SYBR gold. So far, we have established the assay for three different miRNAs and showed that each miRNA binds specifically to its target circular DNA and leads to specific amplification. Moreover, inhibitors can easily be screened for selectivity in the presence of other pre-miRNAs.^[5] The use of unmodified pre-miRNAs reduces the risk of generating assay derived artifacts. Thus, we are optimistic that this assay provides a useful tool for the investigation of miRNA maturation and helps accelerating the search for specific inhibitors of miRNA maturation.

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85 Development of DNA Nanogels and Hydrogels as Injectable Delivery Systems for CpG DNA and Other Nucleic Acid-Based Drugs

DNA can be a building block for constructing a wide variety of unique structures, including but not limited to DNA cubes, DNA origami, DNA polyhedra and DNA hydrogels. In addition to the excellent property as a block, DNA is a biologically active compound that can be and is being used in the treatment of human diseases. One of such compounds is the DNA containing unmethylated CpG dinucleotides with particular flanking sequences (CpG motif), the ligand for Toll-like receptor-9 (TLR9). The ligation of TLR9 triggers the induction of proinflammatory cytokines, and CpG DNA has been developed for the treatment of cancer, allergic diseases, and as adjuvants for vaccines against cancer and infectious diseases. Although the phosphodiester bond of CpG DNA is generally replaced by phosphorothioate one to increase the enzymatic stability, this modification is associated with high levels of cytotoxicity. We hypothesized that the use of DNA nanotechnology can be a new and promising approach to increasing the activity of CpG DNA without changing its chemical structure. Based on this, we have developed a series of DNA assemblies having sticky ends through which the assembled DNA units are connected to each other. The immunostimulatory activity of CpG DNA was significantly increased by the formation of tripod- or tetrapod-like structures. Furthermore, the development of DNA dendrimer by connecting the tripod-like DNA assemblies further increased the activity. Then, large DNA hydrogels were successfully developed using several types of assembled DNA units. We demonstrated that CpG DNA hydrogels activate dendritic cells, continuously release intercalated doxorubicin, an anticancer agent, and was highly effective in inhibiting subcutaneous tumor growth in mice after in vivo administration. Then, we have developed injectable sol-gel transition DNA hydrogels utilizing the difference in salt concentrations between test tubes and biological fluids. We confirmed that the administration of solutions containing DNA assemblies with proper sequences resulted in the formation of DNA hydrogels at the administration sites, such as dermis and nasal cavity. These findings suggest that DNA nanogels (DNA dendrimer) and DNA hydrogels are promising delivery systems for nucleic acid-based drugs, such as CpG DNA, as well as for anticancer agents, antigens and other bioactive compounds.

86 Characterization of Side Reactions During the Annealing of Small Interfering RNA

The process of impurity formation during annealing of siRNA was investigated. siRNAs containing common chemical RNA modifications (2’-O-methyl-, 2’-deoxy-2’-fluoro-, 2’-deoxy-ribose and phosphorothioate linkages) were used to determine major side reactions - such as 2’,3’-isomerisation, strand scission and HF elimination. Individual impurities were characterized using analytical SEC, denaturing and non-denaturing IP-RP-HPLC, DSC and UV-spectrometry. The degradation pathways described in this work can lead to significantly reduced product quality and compromised drug activity. Based on our data, incubation temperature and time as well as the presence of divalent cations are the most critical factors in controlling impurity formation. Without metal-ion catalysts, formation of 2’,5’-isomers was the major side reaction of the two RNA single strands containing 2’-unmodified nucleotides. When Mg²⁺-ions were present, metal-ion-catalyzed degradation of all three investigated RNA strands was observed to a significant extent. For 2’-deoxy-2’-fluoro-modified strands HF-elimination at the modified pyrimidine-nucleotides was the dominant side reaction leading to the formation of 2,2’-O-anhydroribonucleotides and arabinosylnucleotides.All side reactions (isomerisation, elimination and strand scission) were suppressed by duplex formation. Since impurity formation was most pronounced at elevated temperatures, heating of the siRNA solution should be limited to the lowest temperature required for substantial dissociation of the duplex. The optimal annealing temperature may therefore be close to the T_m of the duplex. Divalent cations such as Mg²⁺ catalyze strand scission and are best avoided in annealing solutions. The strong concentration dependency of the T_m in salt-free solutions allows for adjustment of the T_m by changing siRNA concentration.

Taken together, annealing in salt-free solutions allows for lower annealing temperatures, minimizes side reactions and simplifies lyophilisation of the resulting siRNA molecule. Our data provides background to successfully address challenges associated with the manufacture of siRNAs and other nucleic acid therapeutics, such as aptamers, spiegelmers, decoy and antisense oligonucleotides.

87 Synthesis and Properties of Oligonucleotides Having 5’-Terminal 2,2,7-Trimethylguanosine Cap Analogues

The spliceosomal uridine-rich small nuclear ribonucleoprotein (U snRNP) particles are essential cofactors of the spliceosome which assembles on introns of pre-mRNAs and catalyses splicing reactions in the nucleus. The snRNP particles contain snRNAs having a 5’-terminal 2,2,7-trimethylguanosine (TMG) cap structure. The TMG-cap structure is composed of TMG and a triphosphate linkage. TMG-capped snRNAs is produced by hypermethylation of 7-methylguanosine (m⁷G)-capped snRNAs in the cytoplasm and plays an important role in transport of TMG-capped RNAs between the cytoplasm and the nucleus. Moreover, Lührmann and coworkers isolated a transport protein, snurportin 1, that can specifically bind to the 5’-terminal part, TMG^5’pppAmpUmpA, of U1 snRNA from HeLa cytosolic extracts. Although it is unknown if this transport protein can recognize the only part of TMG, there is a potential possibility that oligonucleotides incorporating TMG at their 5’-terminus via a suitable linker can be transported into the nucleus. This possibility would provide a new insight into gene therapy that requires the transportation of antisense, antigene, or decoy oligonucleotide molecules into the nucleus.

In this study, we synthesized oligonucleotides having a TMG-cap structure and the analogues with a pyrophosphate bond or an ethylene glycol linkage as a spacer. The length of linkage in the TMG-cap analogue with a pyrophosphate bond between TMG and the oligonucleotide is shorter than that in an unmodified TMG-cap structure. On the other hand, the length in TMG-cap analogue with an ethylene glycol linkage is longer than that in an unmodified structure. We will also report their biological properties in this paper.

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FIGURE 1.

Structure of TMG-cap and the analogues.

The use of short 8-mer antimiR seed targeting tiny LNAs has recently been shown to provide an efficient means to silence single miRNAs or entire miRNA families. Due to their short length, this class of antimiRs will have many perfect match binding sites in the transcriptome. We wanted to assess whether or not tiny LNA oligos affect transcripts harboring binding sites or proteins translated from such transcripts. To this end, we used Sylamer to perform an unbiased and exhaustive analysis of sequence motifs associated with differentially expressed genes following tiny LNA administration to cell lines or mice. In all experiments with antimiRs, Sylamer analysis showed significant overrepresentation among the up-regulated genes of motifs corresponding to binding sites of the cognate seed. However, sequence motifs corresponding to direct binding sites for the tiny LNAs were not overrepresented. The effect on protein level was analyzed using both small scale iTRAQ analyses of a subset of the samples used for transcription profiling as well as an in vitro luciferase reporter assay of tiny LNAs targeting various parts of the luciferase transcript. The proteomics data also showed up-regulation of predicted miRNA targets following miRNA silencing, while no significant effect was seen on proteins encoded by transcripts harboring a direct binding site. The latter finding was corroborated by data from the luciferase reporter assay. Collectively, our data show that while the transcriptome harbors many perfect match binding sites for short 8-mer oligonucleotides, the presence of high affinity tiny LNAs generally has no effect on the transcripts containing perfect match binding sites or on the levels of protein translated from such transcripts.

89 An RNA Aptamer is a Potential Biomarker for Pancreatic Cancer

Introduction: The symptoms of pancreatic cancer are usually non-specific and late. Existing tumor markers (such as CA19-9 and CEA) are neither sensitive nor specific enough to serve as useful screening tests. Tools for earlier detection could increase the proportion of patients who are cured with surgical resection. Aptamers are oligonucleotide ligands that are identified from large libraries of RNA or DNA molecules using an iterative selection process termed as SELEX (Systematic Evolution of Ligands by Exponential Enrichment). Aptamers bind their specific targets with high affinity and specificity and can be used for the dual purpose of identifying novel biomarkers and detecting them in biological specimens.

Methods: In vitro “positive/negative” SELEX was performed using the conditioned media (the “secretome”) of the MiaPaCa2 human pancreatic ductal adenocarcinoma cell line (positive selection) and the immortalized, non-cancerous human pancreatic ductal cell line HPDE (negative selection). The rationale for “positive/negative” selection was to select for aptamers against molecules that are selectively secreted by cancerous cells but not by normal cells. Standard radioactive filter-based binding assays were used to measure binding.

Results/Conclusion: We have successfully selected several nuclease resistant (2'-fluoro-modified) RNA aptamers that differentially bind the secretome of pancreatic cancer cells compared to non-cancerous cells. One of the aptamers (M9-5) binds pancreatic cancer patient serum with high affinity compared to control serum collected from healthy donors. Using a maximal fraction bound (FB) of 11% as a cutoff, the aptamer discriminated between untreated pancreatic cancer patients (N=19) and healthy volunteers (N=20) with 95% sensitivity and 100% specificity, and the difference in aggregate binding values between the groups is highly significant (P<0.0001, Mann-Whitney U test). In addition, paired serum samples obtained from two patients pre- and post-chemoradiation therapy and pre- and two months post-resection have demonstrated a decrease in M9-5 binding with decreased tumor burden. Notably, serum CA19-9 levels were normal (<40 U/ml) in 62% of cancer patients, and CEA levels were normal (<2.5 ng/ml) in 57% of cancer patients, indicating that our biomarker might be more sensitive than these existing serum biomarkers for pancreatic cancer. For the M9-5 target identification, several steps of biochemical fractionation were performed. At each step of the fractionation, the M9-5 binding activity was assayed to follow the purification scheme. At the end of final purification step the fraction was resolved by gel electrophoresis and after Coomassie staining a ∼20 kDa major polypeptide band was visualized. Cyclophilin B (CypB) was identified upon mass spectrometric analysis of the band. To verify the M9-5 target identification, the biotinylated M9-5 aptamer was used for affinity purification. CypB was detected upon Western blot analysis of the purified proteins. Additionally, to confirm that CypB is the protein target of aptamer M9-5, we measured CypB levels in these same serum samples using ELISA. Similar differential expression between cancer patients and healthy volunteers was observed using the ELISA assay as with the M9-5 binding assay. Also, there was a statistically strong correlation between M9-5 binding and CypB levels by ELISA. The implication of these finding will be further discussed.

90 Endosomal Escape of Liposomal siRNA Via Membrane Fusion and In Vivo Delivery to Tumor

To deliver siRNA to tumor tissue, we have developed liposome type carrier, a multi-functional envelope type nano device (MEND). While PEGylation is the most popular strategy to stabilize liposome in the circulation, reduction of cellular uptake and endosomal escape by PEGylation is a crucial problem. We previously reported that pH-sensitive fusogenic peptide, GALA, modification onto the MEND circumvented this barrier in vitro cultured cell (Sakurai et al., Biol Pharm Bull, 2009). GALA-MEND was destabilized in an acidic compartment endosome because the structure of GALA changed into alpha-helix at low pH (∼5.5), and then GALA on the surface of GALA-MEND induced membrane fusion between MEND and endosome. As a result, encapsulated siRNA was released into cytosol efficiently. Although GALA-modified PEGylated MEND (GALA-MEND) elicits an siRNA delivery efficiency, it was clarified that GALA-MEND intrabenously injected into mice was subject to a rapid clearance from blood circulation. Therefore, we designed a shorter version GALA (shGALA). In this study, we would like to introduce in vivo siRNA delivery with shGALA-modified PEGylated MEND (shGALA-MEND).

shGALA-MEND exhibited a higher knockdown effect (60% reduction, 480 nM) than only PEGylated MEND (PEG-MEND) in vitro. Microscopy and flowcytometry analyses indicated that the result was caused by increasing endosomal escape of siRNA, not enhanced cellular uptake. Because inhibition of acidification of endosome/lysosome compartments by ammonium chloride suppressed siRNA efficiencyof shGALA-MEND, endosomal escape of siRNA by shGALA depended on acidification of endosome. Then, to evaluate the biodistribution of both siRNA and lipid envelope of systemically injected MENDs, we labeled these components with radio isotope. While free siRNA showed a very rapid blood clearance, siRNA assembled with PEG-MEND and shGALA-MEND demonstrated a high blood concentration (>10% ID/mL blood) even 6 hr after injection, and no significant difference was observed between PEG- and shGALA-MEND Next, we investigated the in vivo knockdown and the anti-tumor effect of shGALA-MEND. We administered MENDs into tumor-bearing mice via tail vein 4 times every 24 hr at a dose of 4 mg siRNA/kg body weight, and then measured target gene expression and tumor volume change. A significant mRNA reduction (60%) and inhibition of tumor growth were observed in the group of mice treated with shGALA-MEND. In addition, shGALA-MEND showed no notable somatic and hepatic toxicity. In conclusion, we succeeded in developing an in vivo siRNA delivery system to tumor tissue (Sakurai et al., Biomaterials, in press).

91 In Vivo siRNA Delivery with pH-Sensitive Liposome

Small interfering RNA (siRNA) is the biofunctional nucleic acid inducing RNA interference (RNAi) and is emphasized as the tool for gene therapy. The physicochemical characteristics of siRNA, such as high molecular weight and hydrophilicity prevent passive diffusion across plasma membrane. So, delivery carriers are required which allow siRNA to arrive at target organs and enter target cells. In addition, it is necessary for siRNA to localize cytoplasm which is the place for working and avoid lisosomal compartmentalization. To date, we developed a multifunctional envelope-type nano device (MEND) for siRNA delivery carrier to tumour tissue. The most of conventional liposomal carriers are composed of cationic lipids and have high cationic charge on their surface for the purpose of high affinity for cells but lead to nonspecific interaction with anionic species in the blood and exhibit rapid clearance from blood circulation by the reticulo-endothelial system (RES). This problem can be overcome by using polyethylene glycol (PEG) attached on the particle's surface. Even though, the modification of a lot of PEG actually prolonged circulation time, it dramatically inhibits intracellular trafficking. To solve this ‘PEG dilemma’, we previously developed a cleavable PEG-lipid in response to tumour environment ¹⁾ and a novel endosomal escape peptide device ²⁾ . In the present study, we picked on pH-sensitive lipid, which exhibits neutral at physiological pH and cationic at acidic pH. First, we constructed pH-sensitive MEND by using DODAP, which is known as a pH-sensitive aminolipid but it resulted in lower knockdown activity than conventional cationic MEND using DOTAP, which is known as a cationic lipid. Next, we synthesized a novel pH-sensitive lipid, YSK05 and optimized lipid composition to improve the intracellular trafficking of pH-sensitive MEND. As a result, the optimized pH-sensitive MEND successfully showed 100-fold higher knockdown activity than the conventional cationic MEND. Then, we tried to confirm whether pH-sensitive MEND actually facilitated endosomal escape by depending on endosomal acidification by using ammonium chloride, which buffers lysosomes. Expectively, the knockdown activity of pH-sensitive MEND was dramatically decreased depending on ammonium chloride concentration, which suggested endosomal acidification was important process for pH-sensitive MEND to escaping from endosome. Finally, we evaluated knockdown activity of pH-sensitive MEND in subcutaneous OSRC-II tumour model. Both single local and systemic administration of pH-sensitive MEND-formulated human polo-like kinase 1 (hPLK1) caused reduction in tumour-derived hPLK1 mRNA. RNAi-specific mRNA cleavage products in tumour were found by 5’-RACE PCR, which suggested that mRNA reduction resulted from RNAi and was sequence specific. These results suggest that pH-sensitive MEND is an useful tool for siRNA delivery to tumour tissue and has potent carrier for therapeutics.

92 OTS 2011: PMOs – The Clinical Story

Phosphorodiamidate Morpholino oligomers (PMOs) differ from the older Antisense OligoNucleotides (AONs) by the substitution of a synthetic 6-membered morpholine ring for the ribose and linking the monomeric backbone with phosphorodiamidates rather than phosphorothioates moieties. AVI has pioneered PMO development for 25 years and has tested 5 PMOs in 18 clinical studies in several diseases – neuroinvasive West Nile Virus, Hepatitis C, polycystic kidney disease, cytochrome induction and the lead PMO (AVI-4658, eteplirsen) recently completed phase 2 testing in Duchenne Muscular Dystrophy (DMD) at individual doses of up to 900mg IV (20mg/kg).

Maximum Feasible Doses (MFD) of PMOs were well tolerated in 2 mammalian species over 12 weeks to open an eteplirsen IND allowing evaluation of doses of up to 100mg/kg, and supporting plans for further clinical testing. Recently, 219 clinical doses of eteplirsen were well tolerated in a 12-week of dosing, open label, dose escalation, phase 1b/2 clinical study of eteplirsen with a typical pediatric trial AE profile superimposed on the chronic DMD disease. No significant drug related AEs or SAEs were reported and no AE was deemed probably or definitely drug related. Of particular note, no local injection site reactions were reported. Clinical hematology, coagulation and chemistry (renal function especially) remained stable, and encouraging but variable biological efficacy was demonstrated, especially at the 10 and 20 mg/kg dose levels. A further placebo-controlled, phase 2 study of eteplirsen at higher doses (30 and 50 mg/kg) is due to start dosing in June 2011.

Advanced generation PMO based chemistries are now evolving, based on the neutral and stable PMO backbone, by highly specific positioning of limited positive charges (PMOplus™ chemistry) for viral diseases (Ebola, Marburg and Junin hemorrhagic fevers, Dengue and Influenza), arginine based peptide conjugates (PPMO), or novel internal/external additions (PMO-X™), which have demonstrated better in-vivo cell penetration and potency Preclinical testing with these chemistries is well advanced and clinical testing of the PMOplus chemistry for two phase 1 programs (Ebola and Marburg) began in May 2011.

DNAzymes are synthetic, single-stranded DNA catalysts that can be engineered to bind and cleave the target mRNA of a disease-causing gene. The ability of the 10-23 DNAzyme to specifically cleave RNA with high efficiency under simulated physiological conditions has fuelled expectation that this agent may have useful biological application in a gene inactivation strategy. To explore this potential, we used DNAzymes to validate therapeutic targets for therapeutic approaches to nasopharyngeal carcinomas (NPC).

Epstein-Barr virus (EBV)-encoded latent membrane protein 1 (LMP1) has been known to have oncogenic properties during latent infection in NPC. Genetic manipulation of LMP1 expression may provide a novel strategy for the treatment of NPC. By targeting the LMP1 mRNA, we successfully obtained a phosphorothioate-modified ‘‘10–23’’ DNAzyme namely DZ1, through screening a series of DNAzymes. DZ1 could significantly down-regulate the expression of LMP1 in NPC cells, inhibit cell proliferation, metastasis, promote apoptosis and enhance radiosensitivity of NPC through interfering signal pathways which are abnormally activated by LMP1, including NF-κB, AP-1 and STAT3 signal pathways. In a clinical setting, we, for the first time, demonstrated that intratumoral administration of the DNAzyme in combination with a standard radiotherapy could increase the tumor regression rate in patients. Molecular imaging analysis (MRI) revealed that DNAzyme treatment could negatively impact on the tumor vasculatures. Together, interfering LMP1 signaling pathway could be a promising strategy to target the malignant phenotypes of NPC.

Oral Abstracts

Damha Masad J. McGill University, Montreal, Quebec, Canada Masad J. Damha, PhD, Department of Chemistry, McGill University, 801 Sherbrooke St. West, Montreal, QC, CANADA, H3A-2K6, Telephone: 514-398-7552Email: masad.damha@mcgill.ca

202 Arabinose Modified Antisense and siRNAs: Biological Applications, Structural Considerations, and Delivery Strategies

This presentation will provide an overview of recent work in my laboratory and those of collaborators concerning the properties of oligonucleotides carrying 2′-F sugar modifications, particularly 2′-fluoroarabinonucleic acids (2′-F ANA). We found that the characteristics of 2′-F RNA (C3′-endo) and 2′-fluoroarabinonucleic acid (2′-F ANA; C2′/O4′-endo) make it particularly suitable for the design of highly effective siRNAs. This modification approach also benefits from retention of an A-form dsRNA-like helical conformation. Furthermore, it allows for tuning of duplex thermodynamics, reduced immunostimulation, and increased nuclease resistance without impairment of gene silencing activity against reporter constructs and endogenous genes. In the case of 2′-F ANA:RNA hybrids, a pseudohydrogen bond between the 2′-F and H8 of the nucleobase plays an important role in determining duplex stability, as supported by NMR, molecular modeling, and UV melting experiments. Structural and synthetic aspects of novel fluorinated nucleotide analogues will also be presented.

OPEN IN VIEWER

Applications of both modified antisense and siRNAs towards novel endogenous targets will be described, with a focus on ongoing anticancer applications, e.g., treatment of malignant gliomas. To date, a means for effective delivery of both unmodified and modified antisense and siRNA oligonucleotides to target cells and tissues presents a major challenge impeding the development of therapeutic oligonucleotides. To this end, we will also discuss early advances towards new delivery strategies for these compounds, with and without delivery vehicles to facilitate cell uptake.

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