RNA interference mediated inhibition of platelet-derived endothelial cell growth factor (ECGF1) gene expression using short interfering nucleic acid (siNA)

a technology of platelet-derived endothelial cells and interfering nucleic acids, which is applied in the direction of genetic material ingredients, peptide/protein ingredients, group 5/15 element organic compounds, etc., can solve the problems that the interference activity cannot be assayed, and the modification of kreutzer et al. fails to provide examples or guidance, so as to achieve high specificity and high degree of specificity

Inactive Publication Date: 2005-05-19
SIRNA THERAPEUTICS INC
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Benefits of technology

[0025] In one embodiment, a siNA molecule of the invention has RNAi activity that modulates expression of RNA encoded by an ECGF1 gene. Because ECGF1 genes can share some degree of sequence homology with each other, siNA molecules can be designed to target a class of ECGF1 genes (and associated receptor or ligand genes) or alternately specific ECGF1 genes by selecting sequences that are either shared amongst different ECGF1 targets or alternatively that are unique for a specific ECGF1 target. Therefore, in one embodiment, the siNA molecule can be designed to target conserved regions of ECGF1 RNA sequence having homology between several ECGF1 genes so as to target several ECGF1 genes (e.g., different ECGF1 isoforms, splice variants, mutant genes etc.) with one siNA molecule. In another embodiment, the siNA molecule can be designed to target a sequence that is unique to a specific ECGF1 RNA sequence due to the high degree of specificity that the siNA molecule requires to mediate RNAi activity.
[0026]

Problems solved by technology

However, Kreutzer et al. similarly fails to provide examples or guidance as to what extent these modifications would be tolerated in siRNA molecules.
Further, Parrish et

Method used

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  • RNA interference mediated inhibition of platelet-derived endothelial cell growth factor (ECGF1) gene expression using short interfering nucleic acid (siNA)
  • RNA interference mediated inhibition of platelet-derived endothelial cell growth factor (ECGF1) gene expression using short interfering nucleic acid (siNA)
  • RNA interference mediated inhibition of platelet-derived endothelial cell growth factor (ECGF1) gene expression using short interfering nucleic acid (siNA)

Examples

Experimental program
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Effect test

example 1

Tandem Synthesis of siNA Constructs

[0304] Exemplary siNA 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 siNA synthesis in support of high throughput RNAi screening, and can be readily adapted to multi-column or multi-well synthesis platforms.

[0305] After completing a tandem synthesis of a siNA oligo and its complement 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 siNA 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 behaves as a single mo...

example 2

Identification of Potential siNA Target Sites in any RNA Sequence

[0309] 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 siNA targets having complementarity 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 siNA molecules targeting those sites. Various parameters can be used to determine which sites are the most suitable target sites with...

example 3

Selection of siNA Molecule Target Sites in a RNA

[0310] The following non-limiting steps can be used to carry out the selection of siNAs targeting a given gene sequence or transcript. [0311] 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. [0312] 2. In some instances the siNAs correspond to more than one target sequence; such would be the case for example in targeting different transcripts of the same gene, targeting different transcripts 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 targets, and then the lists are compared to find matching seque...

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Abstract

The present invention concerns methods and reagents useful in modulating platelet-derived endothelial cell growth factor (ECGF1) and/or platelet-derived endothelial cell growth factor receptor (e.g., ECGF1r) gene expression in a variety of applications, including use in therapeutic, diagnostic, target validation, and genomic discovery applications. Specifically, the invention relates to small nucleic acid molecules, such as short interfering nucleic acid (siNA), short interfering RNA (siRNA), double-stranded RNA (dsRNA), micro-RNA (miRNA), and short hairpin RNA (shRNA) molecules capable of mediating RNA interference (RNAi) against ECGF1 and/or ECGF1r gene expression and/or activity. The small nucleic acid molecules are useful in the diagnosis and treatment of cancer, proliferative diseases, macular degeneration, diabetic retinopathy, and any other disease or condition that responds to modulation of ECGF1 and/or ECGF1r expression or activity.

Description

[0001] This application is a continuation of U.S. application Ser. No. 10 / 422,704 filed Apr. 24, 2003, which is a continuation of U.S. application Ser. No. 10 / 417,012 filed Apr. 16, 2003, which is a continuation-in-part of International Patent Application No. PCT / US03 / 05346 filed Feb. 20, 2003, and a continuation-in-part of International Patent Application No. PCT / US03 / 05028 filed Feb. 20, 2003, which claim the benefit of U.S. Provisional Application No. 60 / 358,580 filed Feb. 20, 2002, U.S. Provisional Application No. 60 / 363,124 filed Mar. 11, 2002, U.S. Provisional Application No. 60 / 386,782 filed Jun. 6, 2002, U.S. Provisional Application No. 60 / 406,784 filed Aug. 29, 2002, U.S. Provisional Application No. 60 / 408,378 filed Sep. 5, 2002, U.S. Provisional Application No. 60 / 409,293 filed Sep. 9, 2002, and U.S. Provisional Application No. 60 / 440,129 filed Jan. 15, 2003. All of the applications are incorporated by reference herein in their entireties, including the drawings.FIELD OF T...

Claims

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Application Information

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IPC IPC(8): A61K38/00A61K47/48C12N15/11
CPCA61K9/0019A61K47/48053C12N2330/30A61K47/48092A61K47/48123C07F9/65616C12N15/111C12N2310/14C12N2310/315C12N2310/317C12N2310/321C12N2310/322C12N2310/346C12N2310/351C12N2320/51C12N2310/3521A61K47/544A61K47/549A61K47/554Y02A50/30
Inventor MCSWIGGEN, JAMESCHOWRIRA, BHARAT
Owner SIRNA THERAPEUTICS INC
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