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Compositions and methods for inhibiting gene expression of factor XII

A technology of inhibitory factors and compositions, which can be used in biochemical equipment and methods, inactive components of polymer compounds, drug combinations, etc., and can solve problems such as hemorrhagic effects on life

Inactive Publication Date: 2021-10-12
ARROWHEAD PHARMA INC
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Existing treatments for thromboembolic events target enzymes in the coagulation pathway that are critical for controlling injury-associated blood loss through fibrin formation, and thus, treatment with these agents is critical for potentially life-threatening haemorrhages. to negatively affect

Method used

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  • Compositions and methods for inhibiting gene expression of factor XII
  • Compositions and methods for inhibiting gene expression of factor XII
  • Compositions and methods for inhibiting gene expression of factor XII

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0230] Example 1. RNAi initiator synthesis.

[0231] A) Synthesis. RNAi initiator molecules were synthesized following the phosphoramidite technique on solid phase for oligonucleotide synthesis. Depending on scale, MerMade96E (Bioautomation) or MerMade12 (Bioautomation) was used. In controlled pore glass (CPG, or Synthesis was performed on solid supports from Prime Synthesis, Aston, PA, USA. All DNA, 2'-modified RNA, and UNA phosphoramidites were purchased from Thermo Fisher Scientific (Milwaukee, WI, USA). Specifically, the following 2'-O-methylphosphoramidite was used: (5'-O-dimethoxytrityl-N 6 -(benzoyl)-2′-O-methyl-adenosine-′-O-(2-cyanoethyl-N,N-diisopropylamino) phosphoramidite, 5′-O-di Methoxytrityl-N 4 -(acetyl)-2′-O-methyl-cytidine-3′-O-(2-cyanoethyl-N,N-diisopropylamino)phosphoramidite, (5′-O- Dimethoxy-trityl-N 2 -(isobutyryl)-2′-O-methyl-guanosine-3′-O-(2-cyanoethyl-N,N-diisopropylamino)phosphoramidite and 5′-O- Dimethoxy-trityl-2'-O-methyl-uridine-3'-O...

Embodiment 2

[0235] Example 2. Melittin-like peptide (MLP) delivery polymers.

[0236] A) Melittin-like peptide (MLP) synthesis. All MLPs were prepared using peptide synthesis techniques standard in the art. Independently of the L or D form, the MLP sequence can be retro.

[0237] B) CDM-NAG (N-acetylgalactosamine) synthesis To a solution of CDM (300 mg, 0.16 mmol) in 50 mL of dichloromethane was added oxalyl chloride (2 g, 10 weight equivalents) and dimethylformamide (5 μL) . The reaction was carried out overnight, after which excess oxalyl chloride and dichloromethane were removed by rotary evaporation to yield CDM acid chloride. The acid chloride was dissolved in 1 mL of dichloromethane. To this solution was added 1.1 molar equivalents of (aminoethoxy)ethoxy-2-(acetylamino)-2-deoxy-β-D-galactopyranoside (i.e., aminodiethoxy-ethyl NAG ) and pyridine (200 μL, 1.5 equiv) in 10 mL of dichloromethane. It was then stirred for 1.5 hours. The solvent was then removed and the resulting so...

Embodiment 3

[0258] Example 3. In vitro screening of F12 RNAi initiators.

[0259]A) Human cell background. Candidate sequences identified by in silico analysis as being cross-reactive in humans, nonhuman primates, and mice were screened in vitro as chemically modified canonical siRNAs. Thirty-two in silico identified potential F12 RNAi initiators were synthesized as canonical siRNAs and screened for efficacy in vitro. For screening purposes, the human F12 cDNA sequence (accession number NM_000505) was synthesized and cloned (DNA 2.0, Menlo Park, CA) into a commercially available reporter-based screening plasmid, psiCHECK2 (Promega Corporation, Madison, WI). (Promega)), which produces Renilla luciferase / F12 fusion mRNA. For the efficacy of siRNA in a human background, Hep3B cells, a human hepatocellular carcinoma cell line, were seeded at approximately 10,000 cells / well in 96-well plates. Each of the 32 F12 siRNAs was co-transfected at two concentrations (1 nM and 0.1 nM) with 25 ng F12...

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Abstract

RNA interference (RNAi) initiators for inhibiting the expression of Factor XII (F12) gene by the RNA interference mechanism are disclosed. Also disclosed are pharmaceutical compositions comprising one or more F12 RNAi initiators and one or more excipients capable of delivering the RNAi initiators to hepatocytes. Delivery of the F12 RNAi initiator to hepatocytes in vivo provides inhibition of F12 gene expression and treatment of angioedema, including hereditary angioedema (HAE), venous thromboembolism (VTE), and diseases associated with angioedema.

Description

[0001] This application is a divisional application of Chinese Application No. 201680027422.5 entitled "Composition and Method for Inhibiting Gene Expression of Factor XII". Background technique [0002] Factor XII, a serine protease mainly expressed in the liver and found in the blood, has dual functions in the intrinsic coagulation pathway and in the kinin-kallikrein system. The kinin-kallikrein system plays a role in inflammation, blood pressure control, coagulation, and pain. The active form of factor XII (also known as FXII, FC12, or Hagemann factor) simultaneously cleaves factor XI in the coagulation cascade and prekallikrein in the kallikrein-kallikrein system, yielding the active form of FXI, respectively and kallikrein. [0003] Patients who lost F12 completely did not have bleeding disorders. In addition, mice lacking F12 due to gene knockout were protected from thrombosis (Renne et al., JEM 2005, 202:271-281). The thromboprotective effect of F12 depletion was als...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/113A61K31/713A61K45/06A61K47/42A61K47/54A61P7/10A61P9/00A61P7/02
CPCC12N15/1137A61K31/713A61K45/06A61K47/42A61K47/554A61P7/10A61P9/00A61P7/02C12N2310/14C12N2310/351C12N2320/30C12N2310/3515C12N2310/315C12N2310/322A61K9/0019C12N15/113A61P43/00
Inventor S·B·坎纳D·L·路易斯D·H·维基菲尔德L·埃尔米达
Owner ARROWHEAD PHARMA INC
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