RNA interference-mediated suppression of epithelial sodium channel (ENAC) gene expression using short interfering nucleic acid (SINA)

A technology for interfering nucleic acids and nucleotides, which is applied in the field of RNA interference-mediated inhibition of epithelial sodium channel (ENaC) gene expression using short interfering nucleic acids (siNA), and can solve problems such as reduced RNAi activity

Inactive Publication Date: 2011-12-21
SCHERING AG
View PDF231 Cites 1 Cited by
  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Parrish also reported that incorporation of 5-iodouracil and 3-(aminoallyl)uracil in the antisense strand also resulted in a considerable reduction in RNAi activity

Method used

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
View more

Image

Smart Image Click on the blue labels to locate them in the text.
Viewing Examples
Smart Image
  • RNA interference-mediated suppression of epithelial sodium channel (ENAC) gene expression using short interfering nucleic acid (SINA)
  • RNA interference-mediated suppression of epithelial sodium channel (ENAC) gene expression using short interfering nucleic acid (SINA)
  • RNA interference-mediated suppression of epithelial sodium channel (ENAC) gene expression using short interfering nucleic acid (SINA)

Examples

Experimental program
Comparison scheme
Effect test

preparation example Construction

[0885] Synthetic methods used for RNA comprising certain siNA molecules of the invention follow procedures as described in the following references: Usman et al., 1987, J.Am.Chem.Soc., 109, 7845; Scaringe et al., 1990, Nucleic Acids Res., 18, 5433; and Wincott et al., 1995, Nucleic Acids Res.23, 2677-2684 Wincott et al., 1997, Methods Mol. Bio., 74, 59, and utilize common nucleic acid protection and conjugation Groups such as dimethoxytrityl at the 5'-end, and phosphoramidites at the 3'-end. In a non-limiting example, small-scale synthesis was performed on a 394 Applied Biosystems, Inc. synthesizer using a 0.2 μmol scale protocol with a 7.5-minute coupling step for alkylsilyl-protected nucleotides and a 7.5-minute coupling step for A 2.5 minute coupling step was used for 2'-O-methylated nucleotides. Table 9 summarizes the amounts of reagents and contact times used in the synthesis cycle. Alternatively, synthesis at the 0.2 μmol scale can be performed on a 96-well plate synth...

Embodiment 1

[0992] Example 1: Design, synthesis and identification of siNAs active against ENaCα

[0993] Synthesis of ENaCα siNA

[0994] A series of 64 siNA strands were designed, synthesized and evaluated for efficacy against ENaC. The main criteria for ENaC siNA design were (i) conservation of ENaC across human, mouse and rat isoforms, and (ii) high efficacy score as determined by a proprietary algorithm. The effect of siNA on ENaC protein production and RNA levels was also examined. The siNA sequences designed, synthesized and evaluated for efficacy against ENaC are described in Table 1a (target sequences) and Table 1b (modified sequences).

[0995] Table 1a: ENaCα target sequences, target sites indicated.

[0996]

[0997]

Embodiment 2

[0998] Example 2: Tandem Synthesis of siNA Constructs

[0999] Exemplary siNA molecules of the invention are synthesized in tandem using cleavable linkers, such as succinyl-based linkers. Tandem synthesis as described herein is followed by a single-step purification process that provides RNAi molecules in high yield. This method is highly amenable to siNA synthesis supporting high-throughput RNAi screening and can be easily adapted to multi-column or multi-well synthesis platforms.

[1000] After tandem synthesis of siNA oligonucleotides (oligo) and their complements in which the 5′-terminal dimethoxytrityl (5′-O-DMT) remains intact (trityl-dependent synthesis), the oligonuclear The nucleotides were deprotected as described above. After deprotection, the siNA sequence strands are allowed to hybridize spontaneously. This hybridization produces a duplex in which one strand has retained a 5'-O-DMT group and the complementary strand contains a terminal 5'-hydroxyl. The newly...

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to view more

PUM

PropertyMeasurementUnit
particle sizeaaaaaaaaaa
Login to view more

Abstract

The present invention relates to compounds, compositions and methods for the study, diagnosis and treatment of traits, diseases and conditions in response to regulation of ENaC gene expression and / or activity and / or modulation of ENaC gene expression pathways. In particular, the present invention relates to double-stranded nucleic acid molecules capable of mediating RNA interference (RNAi) against ENaC gene expression or mediating RNA interference (RNAi) against ENaC gene expression, including small nucleic acid molecules such as short interfering nucleic acid (siNA ), short interfering RNA (siRNA), double-stranded RNA (dsRNA), microRNA (miRNA), and short hairpin RNA (shRNA) molecules.

Description

[0001] This application claims U.S. Provisional Application No. 61 / 158,316, filed March 6, 2009, U.S. Provisional Application No. 61 / 118,160, filed November 26, 2008, U.S. Provisional Application No., filed November 26, 2008 61 / 118,157, U.S. Provisional Application No. 61 / 118,150, filed November 26, 2008, U.S. Provisional Application No. 61 / 118,144, filed November 26, 2008. This application claims the benefit of all listed applications, which are hereby incorporated by reference in their entirety, including the drawings. [0002] sequence listing [0003] Pursuant to 37 CFR §1.52(e)(5), the Sequence Listing submitted via EFS is incorporated herein by reference. Sequence Listing text files submitted via EFS include the file "SequenceListing72WPCT" created on November 18, 2009, which is 98,183 bytes in size. field of invention [0004] The present invention relates to compounds, compositions and methods for the study, diagnosis and treatment of traits, diseases and conditions ...

Claims

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to view more

Application Information

Patent Timeline
no application Login to view more
Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/113A61K31/713
CPCC12N2310/321C12N2310/317C12N15/1138C12N2310/315C12N2310/14A61P11/00A61P11/02A61P11/06A61P11/08C12N2310/3521C12N15/113A61K31/713
Inventor V·皮克林W·斯特拉普斯
Owner SCHERING AG
Who we serve
  • R&D Engineer
  • R&D Manager
  • IP Professional
Why Eureka
  • Industry Leading Data Capabilities
  • Powerful AI technology
  • Patent DNA Extraction
Social media
Try Eureka
PatSnap group products

Browse by: Latest US Patents, China's latest patents, Technical Efficacy Thesaurus, Application Domain, Technology Topic.

© 2024 PatSnap. All rights reserved.Legal|Privacy policy|Modern Slavery Act Transparency Statement|Sitemap