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

a technology of double-stranded ribonucleic acid and effector molecules, which is applied in the field of compositions and methods for genespecific inhibition of gene expression by double-stranded ribonucleic acid (dsrna) effector molecules, can solve the problems of complex rules, unable to target sirna molecules, and early attempts to similarly suppress gene expression using long dsrnas in mammalians systems failed, etc.

Inactive Publication Date: 2005-12-15
CITY OF HOPE +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Early attempts to similarly suppress gene expression using long dsRNAs in mammalians 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.

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

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example 1

[0050] This example demonstrates the preparation of double-stranded RNA oligonucleotides

[0051] Oligonucleotide synthesis and purification. 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, Methods Mol Biol 1993, 20:81-114; Wincott et al., Nucleic Acids Res 1995, 23:2677-84). 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.

[0052]...

example 2

[0055] This example demonstrates that dsRNAs having strands that are 25 nucleotides in length or longer have surprisingly increased potency in mammalian systems than known 21-23-mer siRNAs.

[0056] Cell Culture, Transfection, and EGFP Assays. Human embryonic kidney (HEK) 293 cells were grown in DMEM medium supplemented with 10% fetal bovine serum (FBS) (Irvine Scientific, Santa Ana, Calif.). Transfections were done at 90% confluence in 24-well plates using Lipofectamine 2000 (Invitrogen, Carlsbad, Calif.) according to the manufacturer's instructions. Briefly, 50 μl of Opti-MEM media was mixed with nucleic acids, including siRNA duplexes and / or 100-200 ng plasmid pEGFP-C1 (Clontech, Palo Alto, Calif.) for 5 min. Nucleic acids were then mixed with 50 μl of Opti-MEM media that had been pre-mixed with 1.5 μl of Lipofectamine 2000 and incubated at room temperature for 15 min. The lipid—nucleic acid mixtures were added to cells after removal of old media and swirled and then an additional ...

example 3

[0068] This example demonstrates that the use of 25-30 nucleotide RNA duplexes allows gene targeting at a site that could not be effectively targeted using traditional siRNA 21 -mer designs.

[0069] It is currently expected in the art that the majority of 21-mer siRNA duplexes targeted to sites within a given target gene sequence will be ineffective (Holen et al., 2002, Nucleic Acids Res., 30:1757-1766). Consequently, a variety of sites are commonly tested in parallel or pools containing several distinct siRNA duplexes specific to the same target with the hope that one of the reagents will be effective (Ji et al., 2003, FEBS Lett., 552:247-252). To overcome the need to pool or engage in large scale empiric testing, complex design rules and algorithms have been devised to increase the likelihood of obtaining active RNAi effector molecules (Schwarz et al., 2003, Cell, 115:199-208; Khvorova et al., 2003, Cell, 115:209-216; Ui-Tei et al., 2004, Nucleic Acids Res., 32:936-948; Reynolds et...

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Abstract

The invention provides compositions and methods for selectively reducing the expression of a gene product from a desired target gene, as well as treating diseases caused by expression of the gene. The method involves introducing into the environment of a cell an amount of a double-stranded RNA (dsRNA) such that a sufficient portion of the dsRNA can enter the cytoplasm of the cell to cause a reduction in the expression of the target gene. The dsRNA has a first oligonucleotide sequence that is between 26 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 from about 19 to about 23 nucleotides is complementary to a nucleotide sequence of the RNA produced from the target gene.

Description

CROSS-REFERENCE TO RELATED APPLICATION [0001] The present application is related to and claims priority under 35 U.S.C. § 119(e) to U.S. provisional patent application No. 60 / 553,487 filed 15 Mar. 2004, incorporated herein by reference.STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH AND DEVELOPMENT [0002] This invention was made in part with Government support under Grant Numbers AI29329 and HL074704 awarded by the National Institute of Health. The Government may have certain rights in this invention.FIELD OF THE INVENTION [0003] This invention pertains to compositions and methods for gene-specific inhibition of gene expression by double-stranded ribonucleic acid (dsRNA) effector molecules. The compositions and methods are useful in modulating gene expression in a variety of applications, including therapeutic, diagnostic, target validation, and genomic discovery. BACKGROUND OF THE INVENTION [0004] Suppression gene expression by double-stranded RNA (dsRNA) has been demonstrated in ...

Claims

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Application Information

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Patent Type & Authority Applications(United States)
IPC IPC(8): A61K48/00C07H21/02C12N15/11C12N15/113C12N15/85C12Q1/68
CPCC12N15/111C12N15/113C12N2310/14C12N2330/30C12N2320/51C12N2310/51C12N2320/30C12N2310/33A61P1/00A61P1/02A61P1/04A61P1/16A61P3/10A61P5/14A61P7/04A61P7/06A61P11/00A61P11/06A61P15/02A61P17/00A61P17/06A61P19/02A61P21/04A61P25/00A61P25/02A61P27/02A61P27/16A61P29/00A61P31/08A61P31/14A61P35/00A61P35/04A61P37/00A61P37/02A61P37/06A61P37/08C12N2310/50C12N2320/50
Inventor ROSSI, JOHNBEHLKE, MARKKIM, DONGHO
Owner CITY OF HOPE
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