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Immunostimulatory nucleic acid molecules

a nucleic acid and immunomodulatory technology, applied in the field of immunomodulatory nucleic acid molecules, to achieve the effect of increasing the sensitivity of chronic leukemia cells

Inactive Publication Date: 2005-06-09
UNIV OF IOWA RES FOUND +2
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Benefits of technology

The patent is about a new discovery that certain nucleic acids can activate immune cells and change the way they respond to infections. Based on this discovery, the patent describes a new type of nucleic acid that can be used to treat or prevent immune system deficiencies, tumors, and allergic reactions. The nucleic acid molecules have a specific structure that is important for their immunostimulatory activity. The patent also describes a method for delivering the nucleic acid molecules to cells and a method for increasing the sensitivity of leukemia cells to chemotherapy. Overall, the patent presents a new way to use nucleic acids to improve immune function and treat disease.

Problems solved by technology

However, nucleic acids of any size (even many kb long) are immunostimulatory if sufficient immunostimulatory motifs are present, since such larger nucleic acids are degraded into oligonucleotides inside of cells.

Method used

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  • Immunostimulatory nucleic acid molecules

Examples

Experimental program
Comparison scheme
Effect test

example 1

Effects of ODNs on B Cell Total RNA Synthesis and Cell Cycle

[0154] B cells were purified from spleens obtained from 6-12 wk old specific pathogen free DBA / 2 or BXSB mice (bred in the University of Iowa animal care facility; no substantial strain differences were noted) that were depleted of T cells with anti-Thy-1.2 and complement and centrifugation over lympholyte M (Cedarlane Laboratories, Homby, Ontario, Canada) (“B cells”). B cells contained fewer than 1% CD4+ or CD8+ cells. 8×104 B cells were dispensed in triplicate into 96 well microtiter plates in 100 μl RPMI containing 10% FBS (heat inactivated to 65° C. for 30 min.), 50 μM 2-mercaptoethanol, 100 U / ml penicillin, 100 ug / ml streptomycin, and 2 mM L-glutamate. 20 μM ODN were added at the start of culture for 20 h at 37° C., cells pulsed with 1 μCi of 3H uridine, and harvested and counted 4 hr later. Ig secreting B cells were enumerated using the ELISA spot assay after culture of whole spleen cells with ODN at 20 μM for 48 hr....

example 2

Effects of ODN on Production of IgM from B Cells

[0155] Single cell suspensions from the spleens of freshly killed mice were treated with anti-Thyl, anti-CD4, and anti-CD8 and complement by the method of Leibson et al., J. Exp. Med. 154:1681 (1981)). Resting B cells (2% T cell contamination) were isolated from the 63-70% band of a discontinuous Percoll gradient by the procedure of DeFranco et al, J. Exp. Med. 155:1523 (1982). These were cultured as described above in 30 μM ODN or 20 μg / ml LPS for 48 hr. The number of B cells actively secreting IgM was maximal at this time point, as determined by ELIspot assay (Klinman, D. M. et al. J. Immunol. 144:506 (1990)). In that assay, B cells were incubated for 6 hrs on anti-Ig coated microtiter plates. The Ig they produced (>99% IgM) was detected using phosphatase-labelled anti-Ig (Southern Biotechnology Associated, Birmingham, Ala.). The antibodies produced by individual B cells were visualized by addition of BCIP (Sigma Chemical Co., St. L...

example 3

B cell Stimulation by Bacterial DNA

[0156] DBA / 2 B cells were cultured with no DNA or 50 μg / ml of a) Micrococcus lysodeikticus; b) NZB / N mouse spleen; and c) NFS / N mouse spleen genomic DNAs for 48 hours, then pulsed with 3H thymidine for 4 hours prior to cell harvest. Duplicate DNA samples were digested with DNAse I for 30 minutes at 37 C prior to addition to cell cultures. E coli DNA also induced an 8.8 fold increase in the number of IgM secreting B cells by 48 hours using the ELISA-spot assay.

[0157] DBA / 2 B cells were cultured with either no additive, 50 μg / ml LPS or the ODN 1; 1a; 4; or 4a at 20 μM. Cells were cultured and harvested at 4, 8, 24 and 48 hours. BXSB cells were cultured as in Example 1 with 5, 10, 20, 40 or 80 μM of ODN 1; 1a; 4; or 4a or LPS. In this experiment, wells with no ODN had 3833 cpm. Each experiment was performed at least three times with similar results. Standard deviations of the triplicate wells were <5%.

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Abstract

Nucleic acids containing unmethylated CpG dinucleotides and therapeutic utilities based on their ability to stimulate an immune response and to redirect a Th2 response to a Th1 response in a subject are disclosed. Methods for treating atopic diseases, including atopic dermatitis, are disclosed.

Description

Related Applications [0001] This application is a divisional of co-pending U.S. patent application Ser. No. 08 / 738,652, filed Oct. 30, 1996, which is a continuation-in-part of U.S. patent application Ser. No. 08 / 386,063, filed Feb. 7, 1995, now issued as U.S. Pat. No. 6,194,388, which is a continuation-in-part of U.S. patent application Ser. No. 08 / 276,358, filed Jul. 15, 1994, now abandoned.GOVERNMENT SUPPORT [0002] The work resulting in this invention was supported in part by National Institute of Health Grant No. R29-AR42556-01. The U.S. Government may therefore be entitled to certain rights in the invention.BACKGROUND OF THE INVENTION [0003] DNA Binds to Cell Membranes and is Internalized [0004] In the 1970's, several investigators reported the binding of high molecular weight DNA to cell membranes (Lerner, R. A., W. Meinke, and D. A. Goldstein. 1971. “Membrane-associated DNA in the cytoplasm of diploid human lymphocytes”. Proc. Natl. Acad. Sci. USA 68.1212; Agrawal, S. K., R. W...

Claims

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Application Information

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Patent Type & Authority Applications(United States)
IPC IPC(8): A61K9/127A61K31/00C12N15/09A61K31/175A61K31/335A61K31/47A61K31/4706A61K31/70A61K31/7088A61K39/39A61K39/395A61K45/00A61K47/48A61K48/00A61P1/00A61P1/02A61P1/04A61P11/06A61P17/06A61P19/02A61P31/04A61P31/12A61P33/00A61P35/00A61P37/02A61P37/04A61P37/06A61P37/08A61P43/00C07H21/00C07H21/02C07H21/04C07K14/52C12N15/117C12Q1/68
CPCA61K31/00A61K31/4706A61K31/7048A61K31/711A61K31/7125A61K39/00C12Q1/68A61K2039/55561C07H21/00C12N15/117C12N2310/17C12N2310/315A61K39/39A61P1/00A61P1/02A61P1/04A61P1/16A61P11/06A61P17/00A61P17/06A61P19/02A61P31/00A61P31/04A61P31/10A61P31/12A61P33/00A61P35/00A61P37/02A61P37/04A61P37/06A61P37/08A61P43/00A61P7/00Y02A50/30
Inventor KRIEG, ARTHURKLINE, JOELKLINMAN, DENNISSTEINBERG, ALFRED
Owner UNIV OF IOWA RES FOUND
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