Morrissey, DV, Lockridge, JA, Shaw, L, Blanchard, K, Jensen, K, Breen, W et al.. Potent and persistent in vivo anti-HBV activity of chemically modified siRNAs. Nat Biotech 23: 1002-1007

Sirna Therapeutics, Inc., 2950 Wilderness Place, Boulder, Colorado 80301, USA.
Nature Biotechnology (Impact Factor: 41.51). 08/2005; 23(8):1002-7. DOI: 10.1038/nbt1122
Source: PubMed


The efficacy of lipid-encapsulated, chemically modified short interfering RNA (siRNA) targeted to hepatitis B virus (HBV) was examined in an in vivo mouse model of HBV replication. Stabilized siRNA targeted to the HBV RNA was incorporated into a specialized liposome to form a stable nucleic-acid-lipid particle (SNALP) and administered by intravenous injection into mice carrying replicating HBV. The improved efficacy of siRNA-SNALP compared to unformulated siRNA correlates with a longer half-life in plasma and liver. Three daily intravenous injections of 3 mg/kg/day reduced serum HBV DNA >1.0 log(10). The reduction in HBV DNA was specific, dose-dependent and lasted for up to 7 d after dosing. Furthermore, reductions were seen in serum HBV DNA for up to 6 weeks with weekly dosing. The advances demonstrated here, including persistence of in vivo activity, use of lower doses and reduced dosing frequency are important steps in making siRNA a clinically viable therapeutic approach.

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    • "Chemical modification of siRNA designed to inhibit degradation or off-target effects is typically associated with a concomitant reduction in silencing efficacy [33], [34]. However, we previously demonstrated that application of a chemical modification strategy favouring sense strand degradation produces siRNA which retain silencing ability without immune stimulation [13]. "
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    ABSTRACT: Developing siRNA therapeutics poses technical challenges including appropriate molecular design and testing in suitable pre-clinical models. We previously detailed sequence-selection and modification strategies for siRNA candidates targeting STAT6. Here, we describe methodology that evaluates the suitability of candidate siRNA for respiratory administration. Chemically-modified siRNA exhibited similar inhibitory activity (IC50) against STAT6 in vitro compared to unmodified siRNA and apical exposure testing with Caco-2 cell monolayers showed modification was not associated with cellular toxicity. Use of a modified RNA extraction protocol improved the sensitivity of a PCR-based bio-analytical assay (lower limit of siRNA strand quantification = 0.01 pg/µl) which was used to demonstrate that lung distribution profiles for both siRNAs were similar following intra-tracheal administration. However, after 6 hours, modified siRNA was detected in lung tissue at concentrations >1000-fold higher than unmodified siRNA. Evaluation in a rat model of allergic inflammation confirmed the persistence of modified siRNA in vivo, which was detectable in broncho-alveolar lavage (BAL) fluid, BAL cells and lung tissue samples, 72 hours after dosing. Based upon the concept of respiratory allergy as a single airway disease, we considered nasal delivery as a route for respiratory targeting, evaluating an intra-nasal exposure model that involved simple dosing followed by fine dissection of the nasal cavity. Notably, endogenous STAT6 expression was invariant throughout the nasal cavities and modified siRNA persisted for at least 3 days after administration. Coupled with our previous findings showing upregulated expression of inflammatory markers in nasal samples from asthmatics, these findings support the potential of intranasal siRNA delivery. In summary, we demonstrate the successful chemical modification of STAT6 targeting siRNA, which enhanced bio-availability without cellular toxicity or reduced efficacy. We have established a robust, sensitive method for determining siRNA bio-distribution in vivo, and developed a nasal model to aid evaluation. Further work is warranted.
    PLoS ONE 02/2014; 9(2):e90338. DOI:10.1371/journal.pone.0090338 · 3.23 Impact Factor
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    • "Although local delivery of siRNA may be very effective in some settings 6,7,8,9, for many diseases, delivery to disseminated or body-wide targets is required. There have been several steps towards enabling systemic delivery of siRNA, including both improved chemistry to increase serum stability and reduced immunostimulation,4,10 coupled with delivery strategies such as hydrodynamic injection,11,12 liposomes and lipid-based nanoparticles,13,14,15 polyplexes,16,17 ligand-siRNA conjugates,18,19,20 and inorganic particles.21,22 However, there remain major challenges in studying the performance of siRNA given intravenously. "
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    ABSTRACT: Development of RNAi-based therapeutics has the potential to revolutionize treatment options for a range of human diseases. However, as with gene therapy, a major barrier to progress is the lack of methods to achieve and measure efficient delivery for systemic administration. We have developed a positive-readout pharmacodynamic transgenic reporter mouse model allowing noninvasive real-time assessment of siRNA activity. The model combines a luciferase reporter gene under the control of regulatory elements from the lac operon of Escherichia coli. Introduction of siRNA targeting lac repressor results in increased luciferase expression in cells where siRNA is biologically active. Five founder luciferase-expressing and three founder Lac-expressing lines were generated and characterized. Mating of ubiquitously expressing luciferase and lac lines generated progeny in which luciferase expression was significantly reduced compared with the parental line. Administration of isopropyl β-D-1-thiogalactopyranoside either in drinking water or given intraperitoneally increased luciferase expression in eight of the mice examined, which fell rapidly when withdrawn. Intraperitoneal administration of siRNA targeting lac in combination with Lipofectamine 2000 resulted in increased luciferase expression in the liver while control nontargeting siRNA had no effect. We believe a sensitive positive readout pharmacodynamics reporter model will be of use to the research community in RNAi-based vector development.Molecular Therapy-Nucleic Acids (2013) 2, e133; doi:10.1038/mtna.2013.63; published online 19 November 2013.
    Molecular Therapy - Nucleic Acids 11/2013; 2(11):e133. DOI:10.1038/mtna.2013.63 · 4.51 Impact Factor
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    • "To mitigate siRNA-mediated immune stimulation, chemical modifications to the 2¢-OH group in the ribose backbone of siRNAs, including 2¢-O-methyl, 2¢-fluoro, and 2¢-deoxy modifications, and locked nucleic acids (LNAs), have been shown to be effective. Substituting more than 90% of the siRNA nucleotides with a combination of 2¢-O-methyl-, 2¢-fluoro-, and 2¢-deoxy-modified nucleotides can prevent the immunostimulatory response (Morrissey et al., 2005). However, caution must be taken if undertaking extensive chemical modifications to an siRNA as this can significantly impair its silencing activity ( Judge and MacLaclan, 2008). "
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    ABSTRACT: RNA silencing is an established method for investigating gene function and has attracted particular interest due to the potential for generating RNA-based therapeutics. Utilising lentiviral vectors as an efficient delivery system that offers stable, long term expression in post-mitotic cells further enhances the therapeutical applicability of an RNA-based gene therapy for the CNS. In this review we provide an overview of both lentiviral vectors and RNA silencing as well as presenting design considerations for generating lentiviral vectors capable of RNA silencing. We go on to describe the current pre-clinical data regarding lentiviral vector mediated RNA silencing for CNS disorders and discuss the concerns of non-specific side-effects associated with lentiviral vectors and siRNAs and how these might be mitigated.
    Human Gene Therapy Methods 10/2013; 25(1). DOI:10.1089/hgtb.2013.016 · 2.44 Impact Factor
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