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Use of apoptosis-specific elF-5A siRNAs and antisense polynucleotides to inhibit/suppress an inflammatory response

An antisense oligonucleotide and specific technology, applied in the field of inhibiting the expression of apoptosis-specific eIF-5A and DHS, can solve the problem of decreased total protein synthesis

Inactive Publication Date: 2007-08-29
SENESCO TECHNOLOGIES INC
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

(Schnier et al. (1991) Mol. Cell. Biol., 11, 3105-3114; Sasaki et al. (1996) FEBS Lett., 384, 151-154; Park et al. (1998) J. Biol. Chem ., 273, 1677-1683) but deletion of eIF-5A protein in yeast caused only a small decrease in total protein synthesis, indicating that eIF-5A may be necessary for translation of a specific subset of mRNAs rather than for all protein synthesis

Method used

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  • Use of apoptosis-specific elF-5A siRNAs and antisense polynucleotides to inhibit/suppress an inflammatory response
  • Use of apoptosis-specific elF-5A siRNAs and antisense polynucleotides to inhibit/suppress an inflammatory response
  • Use of apoptosis-specific elF-5A siRNAs and antisense polynucleotides to inhibit/suppress an inflammatory response

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preparation example Construction

[0155] Non-natural variants of polynucleotides, antisense oligonucleotides or proteins of eIF-5A or DHS of the invention can be readily prepared using recombinant techniques. These variants include, but are not limited to, those resulting from deletions, additions and substitutions in nucleotide or amino acid sequences. One example of a substitution is a conservative amino acid substitution. This substitution is the replacement of a given amino acid in a protein with another amino acid with similar properties. Typical substitutions that can be considered as conservative substitutions are one or more substitutions between fatty amino acids (Ala, Val, Leu, Ile); exchanges between hydroxyl amino acids Ser and Thr; acidic amino acids Asp and Interchanges between Glu; substitutions between Asn and Gln amino acids; substitutions between basic amino acids Lys and Arg; substitutions between aromatic amino acids Phe and Tyr. For an explanation of which amino acid substitutions may re...

Embodiment 1

[0214] Observation of apoptosis in rat corpus luteum using DNA laddering

[0215] The extent of apoptosis can be determined using a DNA ladder assay. Genomic DNA was isolated from dispersed corpus luteum cells or from isolated corpus luteum tissue using the QIAamp DNA BloodKit (Qiagen) according to the instruction manual. Luteal tissue was removed before induction of apoptosis (treatment with PGF-2α for 1 h) and 1 h and 24 h after induction of apoptosis. Take 500ng genomic DNA, and 0.2μCi[α- 32 P]dCTP, 1mM Tris, 0.5mM EDTA, 3U Klenow enzyme, and 0.2pM each of dATP, dGTP, and dTTP were co-incubated for 30min at room temperature, so as to end-label the isolated DNA. Samples were chromatographed on a 1 ml Sepadex G-50 column to remove unlabeled nucleotides as described by Sambrook et al. Samples were then subjected to Tris-acetate-EDTA (1.8%) gel electrophoresis. The gel was vacuum-dried at room temperature for 30 min, and exposed to X-ray film at -80 °C for 24 h.

[0216] I...

Embodiment 2

[0245] This example illustrates the regulation of apoptosis by apoptosis-specific eIF-5A (apoptosis-specific eIF-5A in the sense direction enhances apoptosis).

[0246] Culture COS-7 cells, RNA isolation

[0247] COS-7 cells, an African green monkey kidney fibroblast-like cell line transformed with a mutant of SV40 (encoding wild T antigen), were used for all transfection experiments. COS-7 cells were cultured with Dulbecco's modified Eagle medium (DMEM) (containing 0.584 g / L L-glutamine, 4.5 g / L glucose and 0.37% sodium bicarbonate). 10% fetal bovine serum (FBS) and 100 units of penicillin / streptomycin were added to the medium. At 37°C, 5% CO 2 and 95% air, the cells were cultured in a humidified environment. Every 3-4 days, the adherent cells were separated with 0.25% trypsin and 1 mM EDTA solution for passage. The detached cells were distributed in a 1:10 split ratio into new dishes containing fresh medium.

[0248] COS-7 cells used for RNA isolation were cultured in 1...

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Abstract

The present invention relates to apoptosis specific eucaryotic initiation factor 5A (eIF-5A), referred to as apoptosis-specific eIF-5A or eIF5-A1, nucleic acids and polypeptides and methods for inhibiting or suppressing apoptosis in cells using antisense nucleotides or siRNAs to inhibit expression of apoptosis-specific eIF-5A. The invention also relates to suppressing or inhibiting expression of pro-inflammatory cytokines or inhibiting activation of NFkB by inhibiting expression of apoptosis-specific eIF-5A.

Description

[0001] related application [0002] This application is a continuation-in-part of U.S. Patent Application Serial No. 10 / 861,980 (filed June 7, 2004), which is a continuation-in-part of U.S. Patent Application Serial No. 10 / 792,893 (filed March 5, 2004) Application, 10 / 792,893 is a continuation-in-part of U.S. Patent Application Serial No. 10 / 383,614 (filed March 10, 2003), 10 / 383,614 is a continuation-in-part of U.S. Patent Application Serial No. 10 / 277,969 (filed October 23, 2002) Continuation-in-Part, 10 / 277,969 is a continuation-in-part of U.S. Patent Application Serial No. 10 / 200,148 (filed July 23, 2002), 10 / 200,148 is a continuation-in-part of U.S. Patent Application Serial No. 10 / 141,647 (filed May 7, 2002 ), 10 / 141,647 is a continuation-in-part of US Patent Application Serial No. 9 / 909,796 (filed July 23, 2001), all of which are incorporated herein by reference. This application also claims U.S. Provisional Patent Application Serial No. 60 / 476,194 (filed on June 6, 2003...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/11A61K35/76C12N15/113
CPCC12N15/113C12N2310/14C12N2310/53C12N2310/111A61P11/00A61P25/28A61P27/06A61P29/00A61P37/00A61P43/00
Inventor J·E·汤普森B·C·加尔顿C·泰勒C·迪纳雷洛L·列兹尼科夫A·布恩M·霍普金斯
Owner SENESCO TECHNOLOGIES INC
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