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

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...

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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] In one embodiment, a siNA molecule of the invention has RNAi activity that modulates expression of RNA encoded by an ECGF1r gene. Because ECGF1r genes can share some degree of sequence homology with each other, siNA molecules can be designed to target a class of ECGF1r genes (and associated receptor or ligand genes) or alternately specific ECGF1r genes by selecting sequences that are either shared amongst different ECGF1r targets or alternatively that are unique for a specific ECGF1r target. Therefore, in one embodiment, the siNA molecule can be designed to target conserved regions of ECGF1r RNA sequence having homology between several ECGF1r genes so as to target several ECGF1r genes (e.g., different ECGF1r 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 ECGF1r RNA sequence due to the high degree of specificity that the siNA molecule requires to mediate RNAi activity.

[0028] In one embodiment, the invention features one or more chemically-modified siNA constructs having specificity for ECGF1 and / or ECGF1r expressing nucleic acid molecules, such as RNA encoding an ECGF1 and / or ECGF1r protein. Non-limiting examples of such chemical modifications include without limitation phosphorothioate internucleotide linkages, 2′-deoxyribonucleotides, 2′-O-methyl ribonucleotides, 2′-deoxy-2′-fluoro ribonucleotides, “universal base” nucleotides, “acyclic” nucleotides, 5-C-methyl nucleotides, and terminal glyceryl and / or inverted deoxy abasic residue incorporation. These chemical modifications, when used in various siNA 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 siNA constructs.

[0050] In a non-limiting example, the introduction of chemically-modified nucleotides into nucleic acid molecules provides a powerful tool in overcoming potential limitations of in vivo stability and bioavailability inherent to native RNA molecules that are delivered exogenously. For example, the use of chemically-modified nucleic acid molecules can enable a lower dose of a particular nucleic acid molecule for a given therapeutic effect since chemically-modified nucleic acid molecules tend to have a longer half-life in serum. Furthermore, certain chemical modifications can improve the bioavailability of nucleic acid molecules by targeting particular cells or tissues and / or improving cellular uptake of the nucleic acid molecule. Therefore, even if the activity of a chemically-modified nucleic acid molecule is reduced as compared to a native nucleic acid molecule, for example, when compared to an all-RNA nucleic acid molecule, the overall activity of the modified nucleic acid molecule can be greater than that of the native molecule due to improved stability and / or delivery of the molecule. Unlike native unmodified siNA, chemically-modified siNA can also minimize the possibility of activating interferon activity in humans.

[0123] In another embodiment, the siNA molecules of the invention are used to target conserved sequences corresponding to a gene family or gene families such as ECGF1 and / or ECGF1r family genes. As such, siNA molecules targeting multiple ECGF1 and / or ECGF1r targets can provide increased therapeutic effect. In addition, siNA can be used to characterize pathways of gene function in a variety of applications. For example, the present invention can be used to inhibit the activity of target gene(s) in a pathway to determine the function of uncharacterized gene(s) in gene function analysis, mRNA function analysis, or translational analysis. The invention can be used to determine potential target gene pathways involved in various diseases and conditions toward pharmaceutical development. The invention can be used to understand pathways of gene expression involved in, for example, the progression and / or maintenance of cancer.

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 al. reported that phosphorothioate modification of more than two residues greatly destabilized the RNAs in vitro such that interference activities could not be assayed.

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