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Compositions and methods for inhibiting gene expression of factor XII

A technology of inhibitory factors and compositions, applied in biochemical equipment and methods, non-effective ingredients of polymer compounds, drug combinations, etc., can solve problems such as the impact of bleeding on life

Active Publication Date: 2018-05-22
ARROWHEAD RES CORP
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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

[0244] Example 1. RNAi initiator synthesis.

[0245] 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-3′-O-(2-cyanoethyl-N,N-diisopropylamino)phosphoramidite, 5′-O- Dimethoxytrityl-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

[0249] Example 2. Melittin-like peptide (MLP) delivery polymers.

[0250] 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.

[0251] 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

[0270] Example 3. In vitro screening of F12 RNAi initiators.

[0271] 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 F1...

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Abstract

RNA interference (RNAi) triggers for inhibiting the expression of Factor XII (F12) gene through the mechanism of RNA interference are described. Pharmaceutical compositions comprising one or more F12RNAi triggers together with one or more excipients capable of delivering the RNAi trigger(s) to a liver cell in vivo are also described. Delivery of the F12 RNAi trigger(s) to liver cells in vivo provides for inhibition of F12 gene expression and treatment of angioedema, including hereditary angioedema (HAE) and venous thromboembolism (VTE), and diseases associated with angioedema.

Description

Background technique [0001] 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. [0002] 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 also observed in F12-inhibitory antibody treated mice, rabbits and primates (Larsson et al., Science TransMed, 2014 6:22ra17). Existing treatments for thromboembolic events targe...

Claims

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Application Information

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IPC IPC(8): C12Q1/68
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 RES CORP
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