Factor xii (hageman factor) (F12), kallikrein b, plasma (fletcher factor) 1 (KLKB1), and kininogen 1 (KNG1) irna compositions and methods of use thereof

A technology of Kallikrein and Flech, applied in the fields of 19D, 2, 19C, acid and table 9, 10, 20, can solve the problems of growth and development of side effects, lack of safety, effectiveness, negative effects, etc.

Active Publication Date: 2018-07-10
ALNYLAM PHARM INC
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Androgens are not suitable for short-term treatment of acute attacks because they take several days to become effective, and they can have significant side effects and can negatively affect growth and development
As a result, androgens are used only for long-term prophylaxis and are typically not administered to pregnant women or children
In addition, current treatments for acute attacks must be administered intravenously multiple times per week or may cause side effects that require dosing or follow-up hospitalization, limiting their routine prophylactic use for long-term management of the disease
Therefore, due to the lack of a safe, effective, and more convenient route of administration and a program for the treatment of acute angioedema attacks and preventive management of recurrent attacks in a large proportion of patients, including pregnant women and children, for patients with HAE There is a need for alternative therapies for test subjects

Method used

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  • Factor xii (hageman factor) (F12), kallikrein b, plasma (fletcher factor) 1 (KLKB1), and kininogen 1 (KNG1) irna compositions and methods of use thereof
  • Factor xii (hageman factor) (F12), kallikrein b, plasma (fletcher factor) 1 (KLKB1), and kininogen 1 (KNG1) irna compositions and methods of use thereof
  • Factor xii (hageman factor) (F12), kallikrein b, plasma (fletcher factor) 1 (KLKB1), and kininogen 1 (KNG1) irna compositions and methods of use thereof

Examples

Experimental program
Comparison scheme
Effect test

example 1

[0700] Example 1: Synthesis of KLKB1 iRNA

[0701] source of reagents

[0702] Herein, if the source of a reagent is not specified, the reagent can be obtained in quality / purity for molecular biology applications from any supplier of reagents for molecular biology.

[0703] transcript

[0704] siRNA design

[0705]For human KLKB1, "kallikrein B, plasma (Flech factor) 1" (REFSeq accession number NM_000892.3, GI: 78191797, GeneID: 3818, SEQ ID NO: 1 and SEQ ID NO.2) and KLKB1 xenologs from virulent species (cynomolgus monkey: RefSeq accession number XM_005556482, GI:544436072; rhesus monkey: RefSeqJU329355, GI:380802470; mouse: RefSeqNM_008455, GI:236465804; rat RefSeqNM_012725, GI:16) A set of siRNAs for targeting is designed using custom R and Python scripts. Human KLKB1 RefSeq mRNA is 2252 bases in length. The rationale and methodology for this set of siRNA designs is as follows: Using a linear model that predicts direct measurements of mRNA knockdown (knockdown), based ...

example 2

[0711] Example 2. In vitro screening of KLKB1 siRNA duplexes

[0712] Cell culture and transfection

[0713] Cos7 cells (ATCC, Manassas, VA) were incubated at 37°C, 5% CO 2 Grow to near confluence in DMEM (ATCC) supplemented with 10% FBS under atmosphere and release from the plate by trypsinization. Luciferase constructs were generated in psiCHECK2 plasmids containing approximately 2.2 kb of human KLKB1 genomic sequence or 2.5 kb of heterologous mouse KLKB1 genomic sequence. Using Lipofectamine 2000 (Invitrogen, Carlsbad CA.cat#11668-019), dual luciferase plasmids were each transfected to 15 x 10 with siRNA. 4 in the cell. For each well of a 96-well plate, 0.2 μl of Lipofectamine was added to 10 ng of plastid vector and single siRNA (Table 3 and Table 4) in 14.8 μL of Opti-MEM, and the complex was left at room temperature for 15 minutes. This mixture was then added to the cells, which were resuspended in 80 μl of fresh complete medium. After incubating the cells for 24 h...

example 3

[0742] Synthesis of Example 3.F12iRNA

[0743] source of reagents

[0744] Herein, if the source of a reagent is not specified, the reagent can be obtained in quality / purity for molecular biology applications from any supplier of reagents for molecular biology.

[0745] transcript

[0746] siRNA design

[0747] For human F12, "Factor XII" (human: NCBI refseqID NM_000505; NCBI GeneID: 2161) and F12 heterologs from virulent species (cynomolgus: XM_005558647; mouse: NM_021489; rat: NM_001014006) are A set of siRNAs targeting one was designed using custom R and Python scripts. Human F12RefSeq mRNA is 2060 bases in length. The rationale and methodology for this set of siRNA designs is as follows: Human F12 mRNA (containing the coding region and 3'UTR) from position 50 to position 2060 (coding region and 3'UTR) for each potential 19mer siRNA predicted potency. This subset of F12 siRNAs was designed to have a perfect or near-perfect fit between humans, cynomolgus monkeys and rh...

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Abstract

The present invention relates to RNAi agents, e.g., double stranded RNAi agents, targeting the Kallikrein B, Plasma (Fletcher Factor) 1 (KLKB1) gene, the Factor XII (Hageman Factor (F12) gene, or theKininogen 1 (KNG1) gene, and methods of using such RNAi agents to inhibit expression of a KLKB1 gene, an F12 gene, and/or a KNG1 gene, and methods of treating subjects having an hereditary angioedema(HAE) and/or a contact activation pathway-associated disorder.

Description

[0001] related application [0002] This application claims U.S. Patent Provisional Application No. 62 / 157,890, filed May 6, 2015, U.S. Patent Provisional Application No. 62 / 260,887, filed November 30, 2015, and December 14, 2015 Priority of U.S. Patent Provisional Application No. 62 / 266,958. The entire contents of each of the aforementioned applications are incorporated herein by reference. [0003] sequence listing [0004] This application contains a Sequence Listing, which has been submitted electronically in ASCII format and is hereby incorporated by reference in its entirety. Said ASCII copy, created on May 3, 2016, is named 121301-03120_SL.txt and is 721,827 bytes in size. Background technique [0005] The coagulation system is essential for hemostasis, producing locally a blood clot formed of a fibrin network and activated platelets in response to vascular injury. Blood coagulation, thrombin generation, and fibrin formation can be initiated by two distinct pathways...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/113A61K31/713
CPCC12N15/113C12N15/1137C12Q1/6883A61K31/713A61K47/549A61K45/06A61P9/12A61P7/10A61P7/02A61P9/10C12N2310/14C12N2310/344C12N2310/321C12N2310/322C12N2310/31C12N2310/3521C12N2310/3533A61P43/00
Inventor A·阿金克G·辛克尔M·迈尔J·巴特勒刘璟璇
Owner ALNYLAM PHARM INC
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