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Methods and compositions for the specific inhibition of gene expression by double-stranded RNA

a technology of double-stranded rna and composition, which is applied in the direction of immunological disorders, metabolism disorders, extracellular fluid disorders, etc., can solve the problems of complex set of rules, sirna molecules are capable of targeting, early attempts to suppress gene expression using long dsrnas in mammalian systems failed, etc., to achieve the best stability, improve stability, and improve stability

Inactive Publication Date: 2009-02-05
CITY OF HOPE +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Benefits of technology

[0034]The compositions and methods have an unanticipated level of potency of the RNAi effect. Although the invention is not intended to be limited by the underlying theory on which it is believed to operate, it is thought that this level of potency and duration of action are caused by the fact the dsRNA serves as a substrate for Dicer which appears to facilitate incorporation of one sequence from the dsRNA into the RISC complex that is directly responsible for destruction of the RNA from the target gene.

Problems solved by technology

Early attempts to suppress gene expression using long dsRNAs in mammalian systems failed due to activation of interferon pathways that do not exist in lower organisms.
Not all siRNA molecules are capable of targeting the destruction of their complementary RNAs in a cell.
As a result, complex sets of rules have been developed for designing RNAi molecules that will be effective.
This technique can lead to artifacts caused by interactions of the siRNA sequences with other cellular RNAs (“off target effects”).
In addition, the duration of the effect of an effective RNAi treatment is limited to about 4 days (Holen et al., 2002).
Immune stimulation constitutes a major class of off-target effects which can dramatically change experimental results and even lead to cell death.
Although cell death can results from immune stimulation, assessing cell viability is not an adequate method to monitor induction of IFN responses.

Method used

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  • Methods and compositions for the specific inhibition of gene expression by double-stranded RNA
  • Methods and compositions for the specific inhibition of gene expression by double-stranded RNA
  • Methods and compositions for the specific inhibition of gene expression by double-stranded RNA

Examples

Experimental program
Comparison scheme
Effect test

example 1

Preparation of Double-Stranded RNA Oligonucleotides

[0130]Oligonucleotide Synthesis and Purification

[0131]RNA oligonucleotides were synthesized using solid phase phosphoramidite chemistry, deprotected and desalted on NAP-5 columns (Amersham Pharmacia Biotech, Piscataway, N.J.) using standard techniques (Damha and Olgivie, 1993; Wincott et al., 1995). The oligomers were purified using ion-exchange high performance liquid chromatography (IE-HPLC) on an Amersham Source 15Q column (1.0 cm×25 cm) (Amersham Pharmacia Biotech, Piscataway, N.J.) using a 15 min step-linear gradient. The gradient varied from 90:10 Buffers A:B to 52:48 Buffers A:B, where Buffer A was 100 mM Tris pH 8.5 and Buffer B was 100 mM Tris pH 8.5, 1 M NaCl. Samples were monitored at 260 nm and peaks corresponding to the full-length oligonucleotide species were collected, pooled, desalted on NAP-5 columns, and lyophilized.

[0132]The purity of each oligomer was determined by capillary electrophoresis (CE) on a Beckman PACE...

example 2

Increased Potency of 25mers

[0136]This example demonstrates that dsRNAs having strands that are 25 nucleotides in length or longer have surprisingly increased potency in mammalian systems than known 21mer to 23mer siRNAs.

[0137]During investigations of the effects of different 5′ and 3′ end structures of dsRNAs made through bacteriophage T7 in vitro transcription (Kim et al., 2004), we observed that some seemed to have greater potency than synthetic 21mer siRNAs directed to the same target site, and that this property seemed to correlate with length. To further explore this phenomenon, we systematically studied the silencing properties of chemically synthesized duplex RNAs of different lengths and designs.

[0138]Cell Culture, Transfection, and EGFP Assays

[0139]HEK 293 cells were split in 24-well plates to 60% confluency in DMEM medium 1 d before transfection. After adding the aliquot of each RNA, 50 μl of Opti Media containing the reporter vectors was added. Next, 50 μl of Opti Media c...

example 3

Dicer Substrates

[0148]This example demonstrates a method for determining whether a dsRNA serves as a substrate for Dicer.

[0149]In Vitro Dicer Cleavage Assays

[0150]RNA duplexes (100 pmol) were incubated in 20 μl of 20 mM Tris pH 8.0, 200 mM NaCl, 2.5 mM MgCl2 with 1 unit of recombinant human Dicer (Stratagene) for 24 h. A 3 μl aliquot of each reaction (15 pmol RNA) was separated in a 15% nondenaturing polyacrylamide gel, stained with GelStar (Ambrex) and visualized using UV excitation.

[0151]Results

[0152]Incubation of 21-bp to 27-bp RNA duplexes with recombinant human Dicer resulted in cleavage of the 23mer, 25mer and 27mer duplexes, but not the 21mer duplex (FIG. 2A). Determinations of relative efficiencies of Dicer cleavage were not possible under the in vitro conditions recommended by the supplier owing to the slow kinetics of this reaction. Aside from the possibility that the dsRNAs longer than 30 bp may need to be preprocessed by Drosha, a micro RNA processing enzyme, to be good ...

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Abstract

The invention is directed to compositions and methods for selectively reducing the expression of a gene product from a desired target gene in a cell, as well as for treating diseases caused by the expression of the gene. More particularly, the invention is directed to compositions that contain double stranded RNA (“dsRNA”), and methods for preparing them, that are capable of reducing the expression of target genes in eukaryotic cells. The dsRNA has a first oligonucleotide sequence that is between 25 and about 30 nucleotides in length and a second oligonucleotide sequence that anneals to the first sequence under biological conditions. In addition, a region of one of the sequences of the dsRNA having a sequence length of at least 19 nucleotides is sufficiently complementary to a nucleotide sequence of the RNA produced from the target gene to trigger the destruction of the target RNA by the RNAi machinery.

Description

CROSS-REFERENCE TO RELATED APPLICATION[0001]The present application is a division of U.S. patent application Ser. No. 11 / 797,216 filed 1 May 2007, which is incorporated herein by reference in its entirety.STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH AND DEVELOPMENT[0002]The present invention was made in part with Government support under Grant Numbers A129329 and HL074704 awarded by the National Institute of Health. The Government may have certain rights in this invention.FIELD OF THE INVENTION[0003]The publications and other materials used herein to illuminate the background of the invention, and in particular, cases to provide additional details respecting the practice, are incorporated by reference, and for convenience are referenced in the following text by author and date and are listed alphabetically by author in the appended bibliography.[0004]The present invention pertains to compositions and methods for gene-specific inhibition of gene expression by double-stranded ribo...

Claims

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Application Information

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Patent Type & Authority Applications(United States)
IPC IPC(8): C12N5/06A61K48/00C07H21/02C12N15/113C12N15/85C12Q1/68
CPCC12N15/111C12N15/113C12N2320/51C12N2310/33C12N2310/14A61P1/04A61P1/16A61P3/10A61P3/12A61P7/00A61P7/06A61P9/00A61P11/06A61P17/00A61P17/02A61P17/06A61P19/02A61P21/04A61P27/02A61P27/16A61P29/00A61P31/14A61P31/18A61P31/20A61P31/22A61P35/00A61P35/02A61P35/04A61P37/06A61P37/08A61P43/00C12N2310/53C12N2310/533C12N2320/35
Inventor ROSSI, JOHN J.BEHLKE, MARK A.KIM, DONGHO
Owner CITY OF HOPE
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