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RNA interference mediated inhibition of proprotein convertase subtilisin Kexin 9 (PCSK9) gene expression using short interfering nucleic acid (siNA)

A nucleotide and double-stranded nucleic acid technology, applied in DNA/RNA fragments, gene therapy, genetic engineering, etc., can solve the problem of RNAi activity reduction

Inactive Publication Date: 2011-07-13
SIRNA THERAPEUTICS INC
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

Parrish also reported that incorporation of 5-iodouracil and 3-(aminoallyl)uracil in the antisense strand also resulted in a considerable reduction in RNAi activity

Method used

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  • RNA interference mediated inhibition of proprotein convertase subtilisin Kexin 9 (PCSK9) gene expression using short interfering nucleic acid (siNA)
  • RNA interference mediated inhibition of proprotein convertase subtilisin Kexin 9 (PCSK9) gene expression using short interfering nucleic acid (siNA)
  • RNA interference mediated inhibition of proprotein convertase subtilisin Kexin 9 (PCSK9) gene expression using short interfering nucleic acid (siNA)

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Experimental program
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preparation example Construction

[0711] The synthetic methods used for RNA comprising certain siNA molecules of the present invention follow the procedures described in the following references: Usman et al., 1987, J. Am. Chem. Soc., 109, 7845; Scaringe et al., 1990, Nucleic Acids Res., 18, 5433; and Wincott et al., 1995, Nucleic Acids Res. 23, 2677-2684 Wincott et al., 1997, Methods Mol. Bio., 74, 59, and utilize common nucleic acid protection and coupling Groups such as dimethoxytrityl at the 5'-end, and phosphoramidite at the 3'-end. In a non-limiting example, the small-scale synthesis was performed on a 394 Applied Biosystems, Inc. synthesizer using a 0.2 μmol scale protocol, a 7.5-minute coupling step for alkylsilyl protected nucleotides and a 2'-O-methylated nucleotides use a 2.5-minute coupling step. Table V summarizes the amount of reagents and contact time used in the synthesis cycle. Alternatively, synthesis on a 0.2 μmol scale can be performed on a 96-well plate synthesizer, such as that produced ...

Embodiment 1

[0824] Example 1: Tandem synthesis of siNA constructs

[0825] Exemplary siNA molecules of the invention are synthesized in tandem using cleavable linkers such as succinyl-based linkers. Tandem synthesis as described herein is followed by a single-step purification process that provides RNAi molecules in high yield. This method is highly compliant with siNA synthesis, supports high-throughput RNAi screening, and can be easily adapted to multi-column or multi-porous synthesis platforms.

[0826] Among them, the 5′-terminal dimethoxytrityl (5′-O-DMT) remains intact (trityl-dependent synthesis) of siNA oligonucleotides (oligo) and their complements. Glycolic acid is deprotected as described above. After deprotection, the siNA sequence strands are allowed to hybridize spontaneously. This hybridization produces a duplex in which one strand has retained the 5'-O-DMT group and the complementary strand contains a terminal 5'-hydroxyl group. The newly formed duplex behaves as a single ...

Embodiment 2

[0830] Example 2: Identification of potential siNA target sites in any RNA sequence

[0831] For example, by using a computer folding algorithm to screen a target RNA target sequence of interest, such as a human mRNA transcript (for example, any sequence mentioned herein by Genbank accession number). In a non-limiting example, sequences derived from databases, such as Genbank genes or RNA gene transcripts, are used to generate siNA targets that have complementarity with the target. Such sequences can be obtained from a database, or can be determined experimentally as known in the art. Known target sites, such as those determined to be effective target sites based on studies using other nucleic acid molecules such as ribozymes or antisense, or those targets known to be related to diseases, traits or conditions, such as those that contain mutations or Those sites that are missing can be used to design siNA molecules that target those sites. Various parameters can be used to dete...

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Abstract

The present invention relates to compounds, compositions, and methods for the study, diagnosis, and treatment of traits, diseases and conditions that respond to the modulation of Proprotein Convertase Subtilisin Kexin 9 (PCSK9) gene expression and / or activity. The present invention is also directed to compounds, compositions, and methods relating to traits, diseases and conditions that respond to the modulation of expression and / or activity of genes involved in Proprotein Convertase Subtilisin Kexin 9 (PCSK9) gene expression pathways or other cellular processes that mediate the maintenance or development of such traits, diseases and conditions. Specifically, the invention relates to double stranded nucleic acid molecules including 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 Proprotein Convertase Subtilisin Kexin 9 (PCSK9) gene expression, including cocktails of such small nucleic acid molecules and lipid nanoparticle (LNP) formulations of such small nucleic acid molecules. The present invention also relates to small nucleic acid molecules, such as siNA, siRNA, and others that can inhibit the function of endogenous RNA molecules, such as endogenous micro-RNA (miRNA) (e.g, miRNA inhibitors) or endogenous short interfering RNA (siRNA), (e.g., siRNA inhibitors) or that can inhibit the function of RISC (e.g., RISC inhibitors), to modulate gene expression by interfering with the regulatory function of such endogenous RNAs or proteins associated with such endogenous RNAs (e.g., RISC), including cocktails of such small nucleic acid molecules and lipid nanoparticle (LNP) formulations of such small nucleic acid molecules. Such small nucleic acid molecules and are useful, for example, in providing compositions to prevent, inhibit, or reduce metabolic diseases.

Description

[0001] This application is a partial continuation application of U.S. Patent Application No. 11 / 487,788 filed on July 17, 2006. The U.S. Patent Application No. 11 / 487,788 is a partial continuation application 11 / 369,108 filed on March 6, 2006. This application is also a partial continuation application of U.S. Patent Application No. 11 / 299,254 filed on December 8, 2005. The U.S. Patent Application No. 11 / 299,254 is U.S. Patent Application No. 11 / 234,730 filed on September 23, 2005. Part of the continuation application of the US Patent Application No. 11 / 234,730 is a part of the continuation application of the US Patent Application No. 11 / 205,646 filed on August 17, 2005. The US Patent Application No. 11 / 205,646 was filed in April 2005. Part of the continuation application of U.S. Patent Application No. 11 / 098,303 filed on August 4, the U.S. Patent Application No. 11 / 098,303 is a part of the continuation application of U.S. Patent Application No. 10 / 923,536 filed on August 20, 200...

Claims

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Application Information

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IPC IPC(8): C12N15/11A61K48/70C12N15/113
CPCC12N2310/14C12N2310/321C12N2310/322C12N15/1137A61P3/04A61P3/06A61P3/10A61P9/00A61P9/04A61P9/06A61P9/10A61P9/12C12N2310/3521
Inventor J·麦斯维根V·贾哈夫C·瓦吉斯
Owner SIRNA THERAPEUTICS INC
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