SIRNA Therapeutics

United States

SIRNA Therapeutics

United States
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Paweletz C.P.,Merck And Co. | Wiener M.C.,Merck And Co. | Bondarenko A.Y.,Rosetta Biosoftware | Bondarenko A.Y.,Microsoft | And 19 more authors.
Journal of Proteome Research | Year: 2010

The rapid identification of protein biomarkers in biofluids is important to drug discovery and development. Here, we describe a general proteomic approach for the discovery and identification of proteins that exhibit a statistically significant difference in abundance in cerebrospinal fluid (CSF) before and after pharmacological intervention. This approach, differential mass spectrometry (dMS), is based on the analysis of full scan mass spectrometry data. The dMS workflow does not require complex mixing and pooling strategies, or isotope labeling techniques. Accordingly, clinical samples can be analyzed individually, allowing the use of longitudinal designs and within-subject data analysis in which each subject acts as its own control. As a proof of concept, we performed multifactorial dMS analyses on CSF samples drawn at 6 time points from n = 6 cisterna magna ported (CMP) rhesus monkeys treated with 2 potent gamma secretase inhibitors (GSI) or comparable vehicle in a 3-way crossover study that included a total of 108 individual CSF samples. Using analysis of variance and statistical filtering on the aligned and normalized LC-MS data sets, we detected 26 features that were significantly altered in CSF by drug treatment. Of those 26 features, which belong to 10 distinct isotopic distributions, 20 were identified by MS/MS as 7 peptides from CD99, a cell surface protein. Six features from the remaining 3 isotopic distributions were not identified. A subsequent analysis showed that the relative abundance of these 26 features showed the same temporal profile as the ELISA measured levels of CSF Abeta 42 peptide, a known pharmacodynamic marker for y-secretase inhibition. These data demonstrate that dMS is a promising approach for the discovery, quantification, and identification of candidate target engagement biomarkers in CSF. © 2010 American Chemical Society.


Haringsma H.J.,Sirna Therapeutics | Li J.J.,Sirna Therapeutics | Soriano F.,Sirna Therapeutics | Kenski D.M.,Sirna Therapeutics | And 2 more authors.
Nucleic Acids Research | Year: 2012

While RNAi has traditionally relied on RNA duplexes, early evaluation of siRNAs demonstrated activity of the guide strand in the absence of the passenger strand. However, these single strands lacked the activity of duplex RNAs. Here, we report the systematic use of chemical modifications to optimize single-strand RNA (ssRNA)-mediated mRNA knockdown. We identify that 2′F ribose modifications coupled with 5′-end phosphorylation vastly improves ssRNA activity both in vitro and in vivo. The impact of specific chemical modifications on ssRNA activity implies an Ago-mediated mechanism but the hallmark mRNA cleavage sites were not observed which suggests ssRNA may operate through a mechanism beyond conventional Ago2 slicer activity. While currently less potent than duplex siRNAs, with additional chemical optimization and alternative routes of delivery, chemically modified ssRNAs could represent a powerful RNAi platform. © 2012 The Author(s).


Chorn G.,Sirna Therapeutics | Zhao L.,Sirna Therapeutics | Sachs A.B.,Sirna Therapeutics | Flanagan W.M.,Sirna Therapeutics | Lim L.P.,Sirna Therapeutics
RNA | Year: 2010

microRNAs are ∼22 nucleotide regulatory RNAs that are processed into duplexes from hairpin structures and incorporated into Argonaute proteins. Here, we show that a nick in the middle of the guide strand of an miRNA sequence allows for seed-based targeting characteristic of miRNA activity. Insertion of an inverted abasic, a dye, or a small gap between the two segments still permits target knockdown. While activity from the seed region of the segmented miRNA is apparent, activity from the 3′ half of the guide strand is impaired, suggesting that an intact guide backbone is required for contribution from the 3′ half. miRNA activity was also observed following nicking of a miRNA precursor. These results illustrate a structural flexibility in miRNA duplexes and may have applications in the design of miRNA mimetics. Published by Cold Spring Harbor Laboratory Press. Copyright © 2010 RNA Society.


PubMed | Sirna Therapeutics
Type: Journal Article | Journal: Molecular pharmacology | Year: 2011

Deeper knowledge of pharmacokinetic and pharmacodynamic (PK/PD) concepts for RNA therapeutics is important to streamline the drug development process and for rigorous selection of best performing drug candidates. Here we characterized the PK/PD relationship for small interfering RNAs (siRNAs) targeting luciferase by examining siRNA concentration in plasma and liver, the temporal RNA-induced silencing complex binding profiles, mRNA reduction, and protein inhibition measured by noninvasive bioluminescent imaging. A dose-dependent and time-related decrease in bioluminescence was detected over 25 days after a single treatment of a lipid nanoparticle-formulated siRNA targeting luciferase messenger RNA. A direct relationship was observed between the degree of in vivo mRNA and protein reduction and the Argonaute2 (Ago2)-bound siRNA fraction but not with the total amount of siRNA found in the liver, suggesting that the Ago2-siRNA complex is the key determinant of target inhibition. These observations were confirmed for an additional siRNA that targets endogenously expressed Sjgren syndrome antigen B (Ssb) mRNA, indicating that our observations are not limited to a transgenic mouse system. Our data provide detailed information of the temporal regulation of siRNA liver delivery, Ago2 loading, mRNA reduction, and protein inhibition that are essential for the rapid and cost-effective clinical development of siRNAs therapeutics.


PubMed | Sirna Therapeutics
Type: Journal Article | Journal: Nucleic acid therapeutics | Year: 2012

Endogenous and exogenous short interfering RNAs (siRNAs) require a 5-phosphate for loading into Ago2 and cleavage of the target mRNA. We applied a synthetic 5-phosphate to siRNA guide strands to evaluate if phosphorylation in vivo is rate limiting for maximal siRNA knockdown and duration. We report, for the first time, an in vivo evaluation of siRNAs with a synthetic 5-phosphate compared to their unphosphorylated versions. siRNAs that contained a 5-phosphate had the same activity in vivo compared with unphosphorylated siRNAs, indicating phosphorylation of an siRNA is not a rate limiting step in vivo.


PubMed | Sirna Therapeutics
Type: Journal Article | Journal: Nucleic acids research | Year: 2012

While RNAi has traditionally relied on RNA duplexes, early evaluation of siRNAs demonstrated activity of the guide strand in the absence of the passenger strand. However, these single strands lacked the activity of duplex RNAs. Here, we report the systematic use of chemical modifications to optimize single-strand RNA (ssRNA)-mediated mRNA knockdown. We identify that 2F ribose modifications coupled with 5-end phosphorylation vastly improves ssRNA activity both in vitro and in vivo. The impact of specific chemical modifications on ssRNA activity implies an Ago-mediated mechanism but the hallmark mRNA cleavage sites were not observed which suggests ssRNA may operate through a mechanism beyond conventional Ago2 slicer activity. While currently less potent than duplex siRNAs, with additional chemical optimization and alternative routes of delivery, chemically modified ssRNAs could represent a powerful RNAi platform.

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