Inhibition of apoptosis-specific eIF-5A ("eIF-5A1") with antisense oligonucleotides and siRNA as anti-inflammatory therapeutics

a technology of apoptosis-specific eucaryotic initiation factor and anti-inflammatory therapy, which is applied in the direction of immunological disorders, extracellular fluid disorders, metabolism disorders, etc., can solve the problems of small decrease in total protein synthesis, binding did not provide protection from ribonucleases, etc., to suppress or suppress the expression of pro-inflammatory cytokines, inhibit or suppress the expression of p53, and increase the expression of bcl-2

Inactive Publication Date: 2006-05-04
SENESCO TECHNOLOGIES INC
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  • Abstract
  • Description
  • Claims
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Benefits of technology

[0021] The present invention relates to apoptosis specific eucaryotic initiation factor 5A (eIF-5A), referred to as “apoptosis specific eIF-5A” or “eIF-5A1.” The present invention also relates to apoptosis-specific eIF-5A 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 present invention relates to a method of delivering siRNA to mammalian lung cells in vivo. The invention also relates to suppressing or inhibiting expression of pro-inflammatory cytokines by inhibiting expression of apoptosis-specific eIF-5A. Further, the present inventio

Problems solved by technology

However, depletion of eIF-5A protein in yeast resulted in only a small decrease in total protein synthesis suggesting that eIF-5A may be required for the translation of specific subsets of mRNA's rather tha

Method used

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  • Inhibition of apoptosis-specific eIF-5A ("eIF-5A1") with antisense oligonucleotides and siRNA as anti-inflammatory therapeutics
  • Inhibition of apoptosis-specific eIF-5A ("eIF-5A1") with antisense oligonucleotides and siRNA as anti-inflammatory therapeutics
  • Inhibition of apoptosis-specific eIF-5A ("eIF-5A1") with antisense oligonucleotides and siRNA as anti-inflammatory therapeutics

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example 1

Visualization of Apoptosis in Rat Corpus Luteum by DNA Laddering

[0195] The degree of apoptosis was determined by DNA laddering. Genomic DNA was isolated from dispersed corpus luteal cells or from excised corpus luteum tissue using the QIAamp DNA Blood Kit (Qiagen) according to the manufacturer's instructions. Corpus luteum tissue was excised before the induction of apoptosis by treatment with PGF-2α, 1 hour and 24 hours after induction of apoptosis. The isolated DNA was end-labeled by incubating 500 ng of DNA with 0.2 μCi[α-32P]dCTP, 1 mM Tris, 0.5 mM EDTA, 3 units of Klenow enzyme, and 0.2 pM each of dATP, dGTP, and dTTP at room temperature for 30 minutes. Unincorporated nucleotides were removed by passing the sample through a 1 ml Sepadex G-50 column according to Sambrook et al. The samples were then resolved by Tris-acetate-EDTA (1.8%) gel electrophoresis. The gel was dried for 30 minutes at room temperature under vacuum and exposed to x-ray film at −80° C. for 24 hours.

[0196]...

example 2

[0217] The present example demonstrates modulation of apoptosis apoptosis-specific eIF-5A (increasing apoptosis with apoptosis-specific eIF-5A in sense orientation)

Culturing of COS-7 Cells and Isolation of RNA

[0218] COS-7, an African green monkey kidney fibroblast-like cell line transformed with a mutant of SV40 that codes for wild-type T antigen, was used for all transfection-based experiments. COS-7 cells were cultured in Dulbecco's Modified Eagle's medium (DMEM) with 0.584 grams per liter of L-glutamine, 4.5 g of glucose per liter, and 0.37% sodium bicarbonate. The culture media was supplemented with 10% fetal bovine serum (FBS) and 100 units of penicillin / streptomycin. The cells were grown at 37° C. in a humidified environment of 5% CO2 and 95% air. The cells were subcultured every 3 to 4 days by detaching the adherent cells with a solution of 0.25% trypsin and 1 mM EDTA. The detached cells were dispensed at a split ratio of 1:10 in a new culture dish with fresh media.

[0219]...

example 3

[0231] The present example demonstrates modulation of apoptosis apoptosis-specific eIF-5A.

[0232] Using the general procedures and methods described in the previous examples, FIG. 23 is a flow chart illustrating the procedure for transient transfection of COS-7 cells, in which cells in serum-free medium were incubated in plasmid DNA in lipofectAMINE for 4 hours, serum was added, and the cells were incubated for a further 40 hours. The cells were then either incubated in regular medium containing serum for a further 48 hours before analysis (i.e. no further treatment), deprived of serum for 48 hours to induce apoptosis before analysis, or treated with actinomycin D for 48 hours to induce apoptosis before analysis.

[0233]FIG. 22 is a Western blot illustrating transient expression of foreign proteins in COS-7 cells following transfection with pHM6. Protein was isolated from COS-7 cells 48 hours after either mock transfection, or transfection with pHM6-LacZ, pHM6-Antisense 3′ rF5A (pHM6...

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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 eIF5A1, 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

RELATED APPLICATIONS [0001] This application is a continuation-in-part of U.S. application Ser. No. 10 / 383,614, filed on Mar. 10, 2003, which is a continuation-in-part of Ser. No. 10 / 277,969, filed Oct. 23, 2002, which is a continuation-in-part of Ser. No. 10 / 200,148, filed on Jul. 23, 2002, which is a continuation-in-part of U.S. application Ser. No. 10 / 141,647, filed May 7, 2002, which is a continuation-in part of U.S. application Ser. No. 9 / 909,796, filed Jul. 23, 2001, all of which are herein incorporated in their entirety. This application also claims priority to U.S. provisional 60 / 476,194 filed on Jun. 6, 2003; U.S. provisional 60 / 504,731 filed on Sep. 22, 2003; U.S. provisional 60 / 528,249 filed on Dec. 10, 2003; U.S. provisional 60 / 557,671 filed on Mar. 21, 2004 and U.S. provisional 60 / (awaited) filed on Jun. 2, 2004, all of which are herein incorporated in their entirety.FIELD OF THE INVENTION [0002] The present invention relates to apoptosis-specific eucaryotic initiation ...

Claims

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Application Information

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IPC IPC(8): A61K48/00C07H21/04C07H21/02C12N15/85A61K31/7088A61K31/713A61P27/00A61P37/00C12NC12N15/113C12N15/12C12Q1/68
CPCA61K31/7088C12N15/113C12N2310/53C12N2310/14C12N2310/11A61P1/00A61P1/02A61P11/06A61P13/12A61P17/04A61P17/06A61P19/00A61P19/02A61P25/00A61P27/00A61P27/02A61P27/06A61P29/00A61P35/00A61P37/00A61P37/02A61P37/08A61P43/00A61P7/00A61P9/00A61P9/10A61P3/10
Inventor THOMPSON, JOHNGALTON, BRUCETAYLOR, CATHERINEDINARELLO, CHARLESREZNIKOV, LEONIDBOONE, ADRIENNEHOPKINS, MARIANNE
Owner SENESCO TECHNOLOGIES INC
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