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PCSK9 iRNA compositions and methods of use thereof

A bonding, AD-60212 technology, applied in PCSK9 iRNA composition and its application field, can solve the problems of myocardial infarction, blood flow occlusion, ruptured clot formation, etc.

Active Publication Date: 2015-08-19
ALNYLAM PHARMA INC
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Alternatively, smaller plaques can rupture and cause clots to form and block blood flow, resulting in, for example, myocardial infarction and / or stroke

Method used

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  • PCSK9 iRNA compositions and methods of use thereof
  • PCSK9 iRNA compositions and methods of use thereof
  • PCSK9 iRNA compositions and methods of use thereof

Examples

Experimental program
Comparison scheme
Effect test

example 1

[0676] Example 1. Synthesis of GalNAc-conjugated oligonucleotides

[0677] A series of siRNA duplexes spanning the PCSK9 mRNA sequence were designed and synthesized using the techniques described above and conjugated with trivalent GalNAc at the 3-terminus of the sense strand. The sequences of these duplexes are shown in Table 1. These same sequences were also synthesized with different nucleotide modifications and conjugated with trivalent GalNAc. The sequences of the modified duplexes are shown in Table 2.

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example 2

[0763] Example 2. In vitro and in vivo screening.

[0764] The potency of a subset of these duplexes in cynomolgus monkey hepatocytes was assessed in a single-dose ad libitum uptake assay. Briefly, primary cynomolgus monkey hepatocytes (PCH) were treated with conjugated modified siRNA duplexes at three concentrations (500 nM, 100 nM and 10 nM). Free uptake assays at 100 nM and 10 nM were performed in duplicate and data are expressed as average message remaining relative to control + / - standard deviation (SD). Screening at 500 nM was performed in a single pass. Table 3 shows the results of these assays.

[0765] Table 3. PCSK9 potency screen by free uptake in primary cynomolgus monkey hepatocytes

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[0773] The efficacy of the modified and conjugated PCSK9 siRNA duplexes was also assessed by transfection assays in three human cell lines. PCSK9 siRNA was transfected into three different cell li...

example 3

[0788] Example 3. Lead optimization.

[0789] Based on the potency assay described in Example 2 above, PCSK9 with various chemical modifications based on the parental sequences of AD-53815 and AD-53806 was evaluated in primary cynomolgus monkey hepatocytes (PCH) in a free uptake assay Potency of siRNA at 200 nM, 20 nM, 2 nM, and 0.2 nM. For all doses except the 0.2 nM dose, determinations were made in duplicate and data were expressed as the average fraction message remaining relative to control. This 0.2 nM dose was determined as a single shot. The results of these assays are shown in Table 6.

[0790] Table 6. Potency screening for lead optimization of AD-53815 and AD-53806 by free uptake in cynomolgus monkey hepatocytes.

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[0798] siRNAs based on the parental sequences of AD-53815 and AD-53806 with various chemical modifications were also screened for in vitro potency by transfection in He...

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Abstract

The invention relates to RNAi agents, e.g., double-stranded RNAi agents, targeting the PCSK9 gene, and methods of using such RNAi agents to inhibit expression of PCSK9 and methods of treating subjects having a lipid disorder, such as a hyperlipidemia.

Description

[0001] related application [0002] This patent application claims U.S. Provisional Application No. 61 / 733,518, filed December 5, 2012, U.S. Provisional Application No. 61 / 793,530, filed March 15, 2013, U.S. Provisional Application No. 61 / 793,530, filed October 4, 2013 61 / 886,916, and priority to U.S. Provisional Application No. 61 / 892,188, filed October 17, 2013. This application is also related to the benefit of US Provisional Application No. 61 / 561,710, filed November 18, 2011. The entire contents of each of the aforementioned provisional patent applications are hereby incorporated by reference. [0003] sequence listing [0004] This application includes a Sequence Listing that has been filed electronically in ASCII format and is hereby incorporated by reference in its entirety. Said ASCII copy, created on October 29, 2013, is named 121301-00420_SL.txt and is 433,512 bytes in size. Background of the invention [0005] Proprotein convertase subtilisin Kexin 9 (PCSK9) is...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/113
CPCC12Y304/21C12N2310/3515C12N2310/315C12N2310/322C12N2310/321C12N15/1137C12N2310/343C12N2310/14A61K47/549A61K31/713A61P3/06A61P43/00C12N2310/3533C12N2310/351C12N2310/312C12N2320/35
Inventor A·博罗多夫斯基R·G·卡兰索塔蒂尔K·菲茨杰拉德M·弗兰克-卡米尼斯基W·奎贝斯M·迈尔K·查里斯S·库奇曼奇M·玛诺哈兰S·米尔斯泰恩
Owner ALNYLAM PHARMA INC
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