Nicked or gapped nucleic acid molecules and uses thereof

a nucleic acid molecule and double-stranded technology, applied in the field of double-stranded nucleic acid molecules, can solve the problems of non-specific silencing of non-targeted genes (referred to as off-target effects) and achieve the effect of maximizing the temperature stability

Inactive Publication Date: 2015-09-17
MARINA BIOTECH INC
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Benefits of technology

The patent text describes a new type of RNA molecule called meroduplex RNA (mdRNA) that can be used to regulate gene expression. This molecule has three strands that can anneal with each other to form double-stranded regions separated by a gap of up to 10 nucleotides. The first strand is complementary to a target RNA, while the second and third strands are each complementary to non-overlapping regions of the first strand. The second and third strands can anneal with each other to form a nick or gap, which can be positioned to maximize thermal stability. The patent also describes the use of specific nucleosides in the mdRNA molecule. Overall, this patent provides a new tool for regulating gene expression and a method for designing specific RNA molecules to achieve this.

Problems solved by technology

Target specific gene silencing can be achieved by exogenously adding siRNA, but non-specific silencing of non-targeted genes (referred to as off-target effects) can be a challenge (see, e.g., Jackson et al., Nat. Biotechnol. 21:635, 2003; Du et al., Nucleic Acids Res.

Method used

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  • Nicked or gapped nucleic acid molecules and uses thereof
  • Nicked or gapped nucleic acid molecules and uses thereof
  • Nicked or gapped nucleic acid molecules and uses thereof

Examples

Experimental program
Comparison scheme
Effect test

example 1

Knockdown of β-Galactosidase Activity by Gapped dsRNA Dicer Substrate

[0194]The activity of a Dicer substrate dsRNA containing a gap in the double-stranded structure in silencing LacZ mRNA as compared to the normal Dicer substrate dsRNA (i.e., not having a gap) was examined.

Nucleotide Sequences of dsRNA and mdRNA Targeting LacZ mRNA

[0195]The nucleic acid sequence of the one or more sense strands, and the antisense strand of the dsRNA and gapped dsRNA (also referred to herein as a meroduplex or mdRNA) are shown below and were synthesized using standard techniques. The RISC activator LacZ dsRNA comprises a 21 nucleotide sense strand and a 21 nucleotide antisense strand, which can anneal to form a double-stranded region of 19 base pairs with a two deoxythymidine overhang on each strand (referred to as 21 / 21 dsRNA).

LacZ dsRNA (21 / 21)—RISC Activator

(SEQ ID NO: 1)Sense5′-CUACACAAAUCAGCGAUUUdTdT-3′(SEQ ID NO: 2)Antisense3′-dTdTGAUGUGUUUAGUCGCUAAA-5′

[0196]The Dicer substrate LacZ dsRNA compr...

example 2

Knockdown of Influenza Gene Expression by Nicked dsRNA

[0200]The activity of a nicked dsRNA (21 / 21) in silencing influenza gene expression as compared to a normal dsRNA (i.e., not having a nick) was examined.

Nucleotide Sequences of dsRNA and mdRNA Targeting Influenza mRNA

[0201]The dsRNA and nicked dsRNA (another form of meroduplex, referred to herein as ndsRNA) are shown below and were synthesized using standard techniques. The RISC activator influenza G1498 dsRNA comprises a 21 nucleotide sense strand and a 21 nucleotide antisense strand, which can anneal to form a double-stranded region of 19 base pairs with a two deoxythymidine overhang on each strand.

G1498-wt dsRNA (21 / 21)

(SEQ ID NO: 7)Sense5′- GGAUCUUAUUUCUUCGGAGdTdT-3′(SEQ ID NO: 8)Antisense3′-dTdTCCUAGAAUAAAGAAGCCUC-5′

[0202]The RISC activator influenza G1498 dsRNA was nicked on the sense strand after nucleotide 11 to produce a ndsRNA having two sense strands of 11 nucleotides (5′-portion, italic) and 10 nucleotides (3′-portion...

example 3

Knockdown Activity of mdRNA Having a Nick in Different Positions

[0208]In this example, the activity of a dicer substrate LacZ dsRNA of Example 1 having a sense strand with a nick at various positions was examined. In addition, a dideoxy nucleotide (i.e., ddG) was incorporated at the 5′-end of the 3′-most strand of a sense sequence having a nick or a single nucleotide gap to determine whether the in vivo ligation of the nicked sense strand is “rescuing” activity. The ddG is not a substrate for ligation. Also examined was the influenza dicer substrate dsRNA of Example 6 having a sense strand with a nick at one of positions 8 to 14. The “p” designation indicates that the 5′-end of the 3′-most strand of the nicked sense influenza sequence was phosphorylated. The “L” designation indicates that the G at position 2 of the 5′-most strand of the nicked sense influenza sequence was substituted for a locked nucleic acid G. The Qneg is a negative control dsRNA.

[0209]The dual fluorescence assay ...

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Abstract

The present disclosure provides meroduplex (nicked or gapped) ribonucleic acid molecules (mdRNA) that decreases or silences target gene expression. An mdRNA of this disclosure comprises at least three strands that combine to form at least two non-overlapping double-stranded regions separated by a nick or gap wherein one strand is complementary to a target gene RNA. In addition, the meroduplex may have one or more modifications or substitutions, such as nucleotide base, sugar, terminal cap structure, internucleotide linkage, or any combination of such modifications. Also provided are methods of decreasing expression of a target gene in a cell or in a subject to treat a disease related to altered expression of a target gene.

Description

TECHNICAL FIELD[0001]The present disclosure provides double-stranded nucleic acid molecules capable of gene silencing and, more specifically, a nicked or gapped double-stranded RNA (dsRNA) comprising at least three strands that decreases expression of a target gene by, for example, RNA interference and uses of such dsRNA to treat or prevent disorders associated with expression of the target gene or genes affected by the target gene.BACKGROUND[0002]RNA interference (RNAi) refers to the cellular process of sequence specific, post-transcriptional gene silencing in animals mediated by small inhibitory nucleic acid molecules, such as a double-stranded RNA (dsRNA) that is homologous to a portion of a targeted messenger RNA (Fire et al., Nature 391:806, 1998; Hamilton et al., Science 286:950-951, 1999). RNAi has been observed in a variety of organisms, including mammalians (Fire et al., Nature 391:806, 1998; Bahramian and Zarbl, Mol. Cell. Biol. 19:274-283, 1999; Wianny and Goetz, Nature C...

Claims

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Application Information

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Patent Type & Authority Applications(United States)
IPC IPC(8): A61K31/713C12N15/11
CPCA61K31/713A61K38/00C12N2310/14C12N2310/315C12N2310/321C12N2310/322C12N2320/51C12N2330/30A61K47/645A61K47/66C12N15/113C12N2310/15C12N15/111A61P29/00A61P31/00A61P35/00C12N2310/3521C12N2310/533C12N2310/3231C12N2310/3513C12N2320/30
Inventor QUAY, STEVEN C.MCSWIGGEN, JAMESVAISH, NARENDRA K.AHMADIAN, MOHAMMAD
Owner MARINA BIOTECH INC
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