Modified Small Interfering RNA Molecules and Methods of Use

a technology of rna interference and small molecules, applied in the field of nucleic acid detection and to the phenomenon of rna interference, or rna interference, can solve the problems of inability to determine the sequence of a mammalian cell type's genome, inability to determine whether every mammalian cell type possesses the necessary machinery, and inability to achieve long-term effects. , to achieve the effect of inhibiting the replication of hcv

Inactive Publication Date: 2012-05-17
NOVARTIS VACCINES & DIAGNOSTICS INC
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, it is not clear that all RNA or DNA sequences of a mammalian cell's genome are susceptible to siRNA.
It is also uncertain that every mammalian cell type possesses the necessary machinery for effectuating gene-specific suppression using siRNA.
Further, siRNA is of limited use for at least two reasons: (a) the transient nature of the suppression effect seen in cells where the siRNA has been administered, and (b) the necessity for chemical synthesis of siRNAs before their use (Tuschl, T., Nature Biotech., 20: 446-448 (2002)).
Also, since siRNAs are unstable in vivo, their long-term effectiveness is limited.
Current therapies for such viral infections are very limited, and tend to have poor response rates.

Method used

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  • Modified Small Interfering RNA Molecules and Methods of Use
  • Modified Small Interfering RNA Molecules and Methods of Use
  • Modified Small Interfering RNA Molecules and Methods of Use

Examples

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example 1

[0107]To test whether siRNA directed to the HCV genome confers intracellular immunity against this human pathogen, a recently developed HCV cell culture systems in human hepatoma cell line, Huh-7, was used. One of the cell lines, 5-2, harbors autonomously replicating subgenomic HCV RNA (Bartenschlager, 7. Virol, 2001). The subgenomic replicon carries firefly luciferase gene, allowing a reporter function assay as a measure of HCV RNA replication (FIG. 5). Owing to cell culture adaptive mutations introduced into the genome (Bart), these 5-2 cells replicate HCV RNA at levels of up to 5×104 virus particles / cell.

[0108]Using T7 transcription, several 21-bp siRNA duplexes against different regions of the 5′-UTR of the HCV genome were made (FIG. 5). Briefly, 2 oligo double-stranded DNA molecules comprising the T7 promoter and the 5′ UTR of HCV being oriented in either the sense direction or the antisense direction were generated. Each oligo DNA was then transcribed in vitro to produce (+) a...

example 2

[0110]The sequence specificity of the siRNA5 response was further tested using additional siRNA duplexes, GL2 and GL3. FIG. 1 shows that GL2 and GL3 differ from each other by 3-nucleotides. Luciferase activity was reduced by 90% in cells transfected with siRNA5 or GL2, but no significant reduction was seen in cells transfected with GL3 (FIG. 7). The luciferase assay was performed using a Luciferase assay system available from Promega Corp. (Madison, Wis.), according to the manufacturer's instructions.

example 3

[0111]Whether or not siRNA5 was toxic to transfected cells also was tested. Toxicity was by measured using an ATPase activity assay. FIG. 8 shows that the siRNA5-induced reduction in HCV replication, as seen in FIG. 6, was not due to cellular toxicity which is attributed to non sequence-specific RNAi. ATPase levels were assayed using an ATPase assay kit from Promega (Madison, Wis.) according to the manufacturer's instructions.

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Abstract

The present invention provides double-stranded RNA molecules that mediate RNA interference in target cells, preferably hepatic cells. The invention also provides double-stranded RNA molecules that are modified to be resistant to nuclease degradation, which inactivates a virus, and more specifically, hepatitis C virus (HCV). The invention also provides a method of using these modified RNA molecules to inactivate virus in mammalian cells and a method of making modified small interfering RNAs (siRNAs) using human Dicer.

Description

BACKGROUND OF THE INVENTION[0001]The present invention relates to the field of nucleic acid detection and to the phenomenon of RNA silencing, or RNA interference (RNAi). RNA silencing constitutes a phenomenon wherein non-coding RNA molecules mediate specific gene suppression in an organism. In nature, the phenomenon protects an organism's genome from foreign, invading nucleic acids such as transposons, trangenes and viral genes.[0002]The introduction of double-stranded RNA (dsRNA) into a cell triggers RNA silencing, which then degrades endogenous mRNA corresponding to the dsRNA. RNA silencing pathways involve a conversion of dsRNA into short interfering RNAs (siRNAs) that direct ribonucleases to homologous mRNA targets (Baulcombe et al., 2001). An enzyme called Dicer processes the dsRNA into siRNAs, which are 20-25 nucleotides long. The siRNAs then assemble into endoribonuclease-containing complexes known as RNA-induced silencing complexes (RISCs). Subsequently, the siRNAs guide the...

Claims

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Application Information

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Patent Type & Authority Applications(United States)
IPC IPC(8): A61K31/713A61P31/14C12N1/21C12N5/10C12N1/19C07H21/02C12N15/63C12N15/09A61K31/7105A61K31/711A61K38/00A61P31/12A61P31/16A61P31/20C12N1/15C12N15/113
CPCA61K31/711A61K38/00C12N15/1131C12N2310/111C12N2310/53C12N2310/322C12N2310/334C12N2310/335C12N2310/3515C12N2310/14A61P1/16A61P11/00A61P31/12A61P31/14A61P31/16A61P31/20A61P43/00Y02A50/30C12N2310/33C12N2320/30
Inventor HAN, JANGSEO, MI-YOUNGHOUGHTON, MICHAEL
Owner NOVARTIS VACCINES & DIAGNOSTICS INC
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