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RNA INTERFERENCE MEDIATED INHIBITION OF GENE EXPRESSION USING CHEMICALLY MODIFIED SHORT INTERFERING NUCLEIC ACID (siNA)

a technology of short interfering nucleic acid and interference, which is applied in the field of synthetic small nucleic acid molecules, can solve the problems of not providing examples of such modified sirna, and similarly failing to show to what extent these modifications are tolerated, so as to improve the various properties of native sirna molecules, modulate rna function and/or, and modulate gene expression

Inactive Publication Date: 2015-04-16
SIRNA THERAPEUTICS INC
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Benefits of technology

The invention relates to compounds that can modify RNA function and gene expression in cells. Specifically, the patent features small nucleic acid molecules like siNA, siRNA, and shRNA that can control gene expression and treat various diseases. Chemical modification of siNA can improve its stability and cellular uptake, allowing for lower dosages and longer half-life in serum. Chemally modified siNA also minimizes the risk of activating interferon activity in humans. Overall, the patent provides useful tools for research and development in the field of RNA therapeutics.

Problems solved by technology

However, Kreutzer and Limmer similarly fail to show to what extent these modifications are tolerated in siRNA molecules nor provide any examples of such modified siRNA.

Method used

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  • RNA INTERFERENCE MEDIATED INHIBITION OF GENE EXPRESSION USING CHEMICALLY MODIFIED SHORT INTERFERING NUCLEIC ACID (siNA)
  • RNA INTERFERENCE MEDIATED INHIBITION OF GENE EXPRESSION USING CHEMICALLY MODIFIED SHORT INTERFERING NUCLEIC ACID (siNA)
  • RNA INTERFERENCE MEDIATED INHIBITION OF GENE EXPRESSION USING CHEMICALLY MODIFIED SHORT INTERFERING NUCLEIC ACID (siNA)

Examples

Experimental program
Comparison scheme
Effect test

example 1

Tandem Synthesis of siNA Constructs

[0584]Exemplary siNA molecules of the invention are synthesized in tandem using a cleavable linker, for example, a succinyl-based linker. Tandem synthesis as described herein is followed by a one-step purification process that provides RNAi molecules in high yield. This approach is highly amenable to siNA synthesis in support of high throughput RNAi screening, and can be readily adapted to multi-column or multi-well synthesis platforms.

[0585]After completing a tandem synthesis of a siNA oligo and its complement in which the 5′-terminal dimethoxytrityl (5′-O-DMT) group remains intact (trityl on synthesis), the oligonucleotides are deprotected as described above. Following deprotection, the siNA sequence strands are allowed to spontaneously hybridize. This hybridization yields a duplex in which one strand has retained the 5′-O-DMT group while the complementary strand comprises a terminal 5′-hydroxyl. The newly formed duplex behaves as a single molecu...

example 2

Serum Stability of Chemically Modified siNA Constructs

[0589]Chemical modifications were introduced into siNA constructs to determine the stability of these constructs compared to native siNA oligonucleotides (containing two thymidine nucleotide overhangs) in human serum. An investigation of the serum stability of RNA duplexes revealed that siNA constructs consisting of all RNA nucleotides containing two thymidine nucleotide overhangs have a half-life in serum of 15 seconds, whereas chemically modified siNA constructs remained stable in serum for 1 to 3 days depending on the extent of modification (see FIG. 3). RNAi stability tests were performed by internally labeling one strand (strand 1) of siNA and duplexing with 1.5× the concentration of the complementary siNA strand (strand 2) (to insure all labeled material was in duplex form). Duplexed siNA constructs were then tested for stability by incubating at a final concentration of 2 μM siNA (strand 2 concentration) in 90% mouse or hu...

example 3

Identification of Potential siNA Target Sites in any RNA Sequence

[0591]The sequence of an RNA target of interest, such as a viral or human mRNA transcript, is screened for target sites, for example by using a computer folding algorithm. In a non-limiting example, the sequence of a gene or RNA gene transcript derived from a database, such as Genbank, is used to generate siNA targets having complementarity to the target. Such sequences can be obtained from a database, or can be determined experimentally as known in the art. Target sites that are known, for example, those target sites determined to be effective target sites based on studies with other nucleic acid molecules, for example ribozymes or antisense, or those targets known to be associated with a disease or condition such as those sites containing mutations or deletions, can be used to design siNA molecules targeting those sites. Various parameters can be used to determine which sites are the most suitable target sites within...

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Abstract

The present invention concerns methods and reagents useful in modulating gene expression in a variety of applications, including use in therapeutic, diagnostic, target validation, and genomic discovery applications. Specifically, the invention relates to synthetic chemically modified small nucleic acid molecules, such as short interfering nucleic acid (siNA), short interfering RNA (siRNA), double-stranded RNA (dsRNA), micro-RNA (miRNA), and short hairpin RNA (shRNA) molecules capable of mediating RNA interference (RNAi) against target nucleic acid sequences. The small nucleic acid molecules are useful in the treatment of any disease or condition that responds to modulation of gene expression or activity in a cell, tissue, or organism.

Description

[0001]This application is a continuation of U.S. patent application Ser. No. 10 / 444,853, filed May 23, 2003, a continuation-in-part of U.S. patent application Ser. No. 10 / 422,704, filed Apr. 24, 2003, a continuation-in-part of U.S. patent application Ser. No. 10 / 417,012, filed Apr. 16, 2003, a continuation-in-part of International Patent Application No. PCT / US03 / 05346, filed Feb. 20, 2003, and a continuation-in-part of International Patent Application No. PCT / US03 / 05028, filed Feb. 20, 2003, all of which claim the benefit of U.S. Provisional Application No. 60 / 358,580 filed Feb. 20, 2002, U.S. Provisional Application No. 60 / 363,124 filed Mar. 11, 2002, U.S. Provisional Application No. 60 / 386,782 filed Jun. 6, 2002, U.S. Provisional Application No. 60 / 406,784 filed Aug. 29, 2002, U.S. Provisional Application No. 60 / 408,378 filed Sep. 5, 2002, U.S. Provisional Application No. 60 / 409,293 filed Sep. 9, 2002, and U.S. Provisional Application No. 60 / 440,129 filed Jan. 15, 2003. This appli...

Claims

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Application Information

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Patent Type & Authority Applications(United States)
IPC IPC(8): C12N15/113C12N15/09A61K31/7088A61K31/7105A61K31/711A61K31/7115A61K31/712A61K31/7125A61K31/713A61K38/00A61K47/48A61K48/00A61P1/00A61P1/04A61P1/16A61P3/00A61P3/10A61P11/06A61P13/08A61P13/10A61P13/12A61P17/00A61P17/02A61P19/00A61P19/02A61P21/00A61P25/00A61P25/02A61P25/28A61P27/02A61P27/16A61P29/00A61P31/00A61P31/04A61P31/10A61P31/12A61P31/14A61P31/16A61P31/18A61P31/20A61P31/22A61P35/00A61P35/02A61P37/00A61P37/04A61P37/06A61P37/08A61P43/00C07H21/02C07H21/04C12N15/11C12N15/82
CPCC12N15/113C12N15/1131C12N15/1133C12N15/1132C12N2320/30C12N2310/14C12N2320/51C12N2310/531C12N15/1138C12N2310/315C12N2310/317C12N2310/331C12N2310/351C12N2310/3515C12N2320/11C12N2330/30A61K38/00C07H21/02C12N15/111C12N15/8218C12N2310/111C12N2310/318C12N2310/321C12N2310/322C12N2310/332C12N2310/346C12N2310/53A61K47/54A61P1/00A61P1/04A61P1/16A61P11/06A61P13/08A61P13/10A61P13/12A61P17/00A61P17/02A61P19/00A61P19/02A61P21/00A61P25/00A61P25/02A61P25/28A61P27/02A61P27/16A61P29/00A61P3/00A61P31/00A61P31/04A61P31/10A61P31/12A61P31/14A61P31/16A61P31/18A61P31/20A61P31/22A61P35/00A61P35/02A61P37/00A61P37/04A61P37/06A61P37/08A61P43/00A61P3/10C12N2310/3521C12N2310/3533
Inventor MCSWIGGEN, JAMESCHOWRIRA, BHARATBEIGELMAN, LEONIDMACEJAK, DENNISZINNEN, SHAWNPAVCO, PAMELAHAEBERLI, PETERMORRISSEY, DAVIDFOSNAUGH, KATHYJAMISON, SHARON F.USMAN, NASSIMTHOMPSON, JAMESVARGEESE, CHANDRAWANG, WEIMINCHEN, TONGQIANVAISH, NARENDRA K.
Owner SIRNA THERAPEUTICS INC
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