RNA interference mediated inhibition of NOGO and NOGO receptor gene expression using short interfering RNA

a technology of rna interference and nogo receptor, which is applied in the direction of genetic material ingredients, biochemistry apparatus and processes, peptides/proteins, etc., can solve the problems of nogo dependent inhibition of axonal growth in these cells, failure to show to what extent these modifications are tolerated, etc., to increase the serum stability of modified sirna constructs, the effect of preserving rna activity in cells

Inactive Publication Date: 2005-11-24
RIBOZYME PHARMA INC
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Benefits of technology

[0031] In one embodiment, the invention features chemically modified siRNA constructs having specificity for NOGO and / or NOGOr expressing nucleic acid molecules. Non-limiting examples of such chemical modifications include without limitation phosphorothioate internucleotide linkages, 2′-O-methyl ribonucleotides, 2′-deoxy-2′-fluoro ribonucleotides, “universal base” nucleotides, 5-C-methyl nucleotides, and inverted deoxyabasic residue incorporation. These chemical modifications, when used in various siRNA constructs, are shown to preserve RNAi activity in cells while at the same time, dramatically increasing the serum stability of these compounds. Furthermore, contrary to the data published by Parrish et al., supra, applicant demonstrates that multiple (greater than one) phosphorothioate substitutions are well tolerated and confer substantial increases in serum stability for modified siRNA constructs. Chemical modifications of the siRNA constructs can also be used to improve the stability of the interaction with the target RNA sequence and to improve nuclease resistance.
[0115] The siRNA molecules of the invention represent a novel therapeutic approach to treat a variety of pathologic indications, including CNS injury and cerebrovascular accident (CVA, stroke), Alzheimer's disease, dementia, multiple sclerosis (MS), chemotherapy-induced neuropathy, amyotrophic lateral sclerosis (ALS), Parkinson's disease, ataxia, Huntington's disease, Creutzfeldt-Jakob disease, muscular dystrophy, and / or other neurodegenerative disease states which respond to the modulation of NOGO and NOGO receptor expression and / or any other diseases or conditions that are related to the levels of NOGO and / or NOGOr in a cell or tissue, alone or in combination with other therapies. The reduction of NOGO and / or NOGOr expression (specifically NOGO and / or NOGOr RNA levels) and thus reduction in the level of the respective protein relieves, to some extent, the symptoms of the disease or condition.

Problems solved by technology

However, Kreutzer and Limmer similarly fail to show to what extent these modifications are tolerated in siRNA molecules nor do they provide any examples of such modified siRNA.
Furthermore, the expression of the NOGO-66 receptor in neurons that are NOGO insensitive results in NOGO dependent inhibition of axonal growth in these cells.

Method used

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  • RNA interference mediated inhibition of NOGO and NOGO receptor gene expression using short interfering RNA
  • RNA interference mediated inhibition of NOGO and NOGO receptor gene expression using short interfering RNA
  • RNA interference mediated inhibition of NOGO and NOGO receptor gene expression using short interfering RNA

Examples

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example 1

Tandem Synthesis of siRNA Constructs

[0218] Exemplary siRNA 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 siRNA synthesis in support of high throughput RNAi screening, and can be readily adapted to multi-column or multi-well synthesis platforms.

[0219] After completing a tandem synthesis of an siRNA oligo and its compliment 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 siRNA 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 to behaves as a s...

example 2

Identification of Potential siRNA Target Sites in Any RNA Sequence

[0223] 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 siRNA targets having complimentarity 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 siRNA molecules targeting those sites as well. Various parameters can be used to determine which sites are the most suitable target...

example 3

Selection of siRNA Molecule Target Sites in a RNA

[0224] The following non-limiting steps can be used to carry out the selection of siRNAs targeting a given gene sequence or transcipt.

[0225] 1. The target sequence is parsed in silico into a list of all fragments or subsequences of a particular length, for example 23 nucleotide fragments, contained within the target sequence. This step is typically carried out using a custom Perl script, but commercial sequence analysis programs such as Oligo, MacVector, or the GCG Wisconsin Package can be employed as well.

[0226] 2. In some instances the siRNAs correspond to more than one target sequence; such would be the case for example in targeting many different strains of a viral sequence, for targeting different transcipts of the same gene, targeting different transcipts of more than one gene, or for targeting both the human gene and an animal homolog. In this case, a subsequence list of a particular length is generated for each of the targe...

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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 associated with Alzheimer's disease. Specifically, the invention relates to small interfering RNA (siRNA) molecules capable of mediating RNA interference (RNAi) against NOGO and NOGO receptor (NOGOr) polypeptide and polynucleotide targets.

Description

BACKGROUND OF THE INVENTION [0001] The present invention concerns methods and reagents useful in modulating NOGO and NOGO receptor gene expression in a variety of applications, including use in therapeutic, diagnostic, target validation, and genomic discovery applications. Specifically, the invention relates to short interfering nucleic acid molecules capable of mediating RNA interference (RNAi) against beta-secretase NOGO and / or NOGO receptor (NOGOr) expression. [0002] The following is a discussion of relevant art pertaining to RNAi. The discussion is provided only for understanding of the invention that follows. The summary is not an admission that any of the work described below is prior art to the claimed invention. [0003] RNA interference refers to the process of sequence-specific post transcriptional gene silencing in animals mediated by short interfering RNAs (siRNA) (Fire et al, 1998, Nature, 391, 806). The corresponding process in plants is commonly referred to as post tran...

Claims

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Application Information

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Patent Type & Authority Applications(United States)
IPC IPC(8): A61K38/00A61K48/00C12N5/00C12N5/02C12N15/113
CPCA61K38/00C12N15/113C12N2310/346C12N15/1137C12N15/1138C12N2310/12C12N2310/121C12N2310/13C12N2310/14C12N2310/18C12N2310/315C12N2310/317C12N2310/321C12N2310/332C12N2310/3521
Inventor MCSWIGGEN, JAMES
Owner RIBOZYME PHARMA INC
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