WO1995032628A1

WO1995032628A1 - Oligonucleotide inhibitors of cytokine production and tumors responsive to cytokines

Info

Publication number: WO1995032628A1
Application number: PCT/US1995/006987
Authority: WO
Inventors: Robert Francis Rando; June B. Santon
Original assignee: Triplex Pharmaceutical Corporation
Priority date: 1994-05-31
Filing date: 1995-05-31
Publication date: 1995-12-07

Abstract

A method for inhibiting cytokine production in cells, TNF-α protein production in cells and growth of TNF-α responsive cells is provided. The method includes the step of administering a therapeutic dose of an oligonucleotide. The oligonucleotide has a composition of guanosine sufficient to produce a three-dimensional structure stabilized by guanosine tetrads or has a composition of at least two runs of at least two contiguous guanosines. In other variations of the method, the composition of guanosine and thymidine is at least 70 %. Also disclosed are specific oligonucleotides useful in inhibition of TNF-α.

Description

"OLIGONUCLEOTIDE INHIBITORS OF CYTOKINE PRODUCTION AND TUMOR RESPONSIVE TO CYTOKINES"

FIELD OF THE INVENTION This invention relates to the method of inhibiting cytokine production by inhibition of cytokine gene expression or reduction in bioactive cytokine levels. This application is further addressed to the inhibition of tumor cell growth in those tumors which are responsive to cytokine factors.

BACKGROUND A variety of antiviral oligonucleotides have been used as antisense agents. These oligonucleotides have been designed against virus or cellular targets and claim inhibition of transcription or translation by an antisense or triple helix mechanism. It is known that an oligodeoxycytodine (poly dC) can be used against HIV-1 and is effective, especially when the oligonucleotide is modified by the addition of sulfur to the internucleoside linkages

(phosphorothioate). In addition to HIV-1, non-antisense oligonucleotides such as poly dC can be used to directly inhibit a variety of other viral diseases. It appears that the poly dC directly inhibits viral reverse transcriptase. Other mechanisms of viral disease inhibition by poly dC can include interaction with AMV reverse transcriptase as well as Pol I (Klenow fragment) and Pol

(alpha). It is also known that diphosphorothioate molecules which are antisense specific, random nucleotide combinations or poly dC are effective against HIV-1. It is believed that these molecules all work by direct inhibition of the HIV-1 reverse transcriptase. These mechanisms are all proposed since the actual mode of action is still unknown. Other potential mechanisms of anti-viral action of oligonucleotides include promotion of RNAse H activity and inhibition of reverse transcriptase initiating cDNA synthesis. It is believed that the phosphorothioate derivatives of polynucleotides inhibit human DNA polymerases and RNAse A. Although when a base was removed from an antisense polynucleotide to form an abasic site, the compound did not lose its activity. This suggests that there is no need for the formation of an RNA:DNA antisense mediated hybrid for the oligonucleotide's activity. There are also some unexpected results showing that an oligonucleotide designed to inhibit HIV-1 via the formation of a triple helix with HIV-1 duplex DNA had in fact formed a duplex with HIV-1 RNA, and as such inhibited the virus by becoming a substitute for RNase H. This is an alternative mechanism of action of an oligonucleotide designed to form a triple helix.

The present invention is designed to reduce cytokine levels in vivo which help produce pathophysiologic conditions by the use of oligonucleotides. It provides a new way of inhibiting the production of the cytokine protein and thus can be used for the treatment of a variety of pathophysiologic conditions which are related to cytokine levels in vivo.

SUMMARY OF THE INVENTION

An object of the present invention is provision of a method for inhibiting either cytokine production or bioactivity.

A further object of the present invention is a method of inhibiting TNF-α protein production or bioactivity.

An additional object of the present invention is a method of inhibiting lipopolysaccharide induced TNF-α protein either production or bioactivity.

A further object of the present invention is a method of inhibiting the growth of TNF-α responsive tumor cells. An additional object of the present invention is provision of novel oligonucleotides for the inhibition of the TNF alpha gene.

Thus, in accomplishing the foregoing objects, there is provided in accordance with one aspect of the present invention a method for inhibiting cytokine production in cells comprising the step of administering a therapeutic dose of an oligonucleotide, said oligonucleotide having a composition of guanosine sufficient to produce a three-dimensional structure stabilized by guanosine tetrads or at least two segments (runs) of at least two contiguous guanosines. Said oligonucleotide is comprised of at least 25% guanosine or guanosine derivatives.

In specific embodiments of this invention, TNF-α protein production or activity, lipopolysaccharide (or other mitogens) induction of TNF-α protein production or activity and growth of tumor cells responsive to TNF alpha can be inhibited.

Additional specific embodiments include the oligonucleotides useful in the inhibition of TNF-α, said oligonucleotides having at least two segments (runs) of at least two contiguous guanosines. In other embodiments of this invention, oligonucleotides capable of the inhibition of TNF-α are comprised of at least 70% guanosine and thymidine and/or naturally-occurring or synthetic derivatives of guanosine and thymidine.

Other and further objects, features and advantages will be apparent and the invention more readily understood from a reading of the following specification and by reference to the accompanying drawings forming a part thereof, wherein the examples of the presently preferred embodiments of the invention are given for the purposes of disclosure.

DESCRIPTION OF THE DRAWINGS Figure 1 is a representation of the levels of TNF-α four hours post stimulation of THP-1 cells with 1 ug/ml LPS and treated with the triplex oligonucleotides of the present invention.

Figure 2 shows the TNF-α levels four hours post stimulation of THP-1 cells with 0.1 ug/ml LPS and treated with triplex oligonucleotides. Figure 3 is a graphic representation of inhibition of TNF-α responsive glioblastoma cell lines treated with TFO.

The drawings and figures are not necessarily to scale and certain features mentioned may be exaggerated in scale or shown in schematic form in the interest of clarity and conciseness. DETAILED DESCRIPTION OF THE INVENTION

It will be readily apparent to one skilled in the art that various substitutions and modifications may be made to the invention disclosed herein without departing from the scope and the spirit of the invention. The term "TFO" or "triplex-forming oligonucleotide" as used herein refer to the oligonucleotides of the present invention. Although these oligonucleotides were designed according to the methods and procedures described in Hogan, et al., U.S. Serial No. 5,176,996, issued January 5, 1993. It is believed that these molecules do not act by forming a triplex in the present invention.

The term "oligonucleotides" as used herein is defined as a molecule comprising two or more deoxyribonucleotides or ribonucleotides, preferably more than ten. The exact size will depend on many factors, including the specificity and binding affinity to target ligands. In referring to "bases" herein, the term includes both deoxyribonucleic acids and ribonucleic acids.

The following abbreviations are used: "A" refers to adenine as well to deoxyribose derivatives, T refers to thymine, "U" refers to the ribose derivative Uracil, "G" refers to guanine as well as its deoxyribose derivatives, "C" refers to cytokine as well as its deoxyribose derivatives, "N" in sequences refers to guanosine analogs, "I" refers to inosine and "F" refers to 5 fluorodeoxy uridine.

As used herein, TNF-α refers to the tumor necrosis factor α. The term "inhibition of the growth of cells" is meant to include partial and total growth inhibition and include decreases in the rate of proliferation or growth of the cells. The term "inhibition" of a gene or protein production is meant to include partial and total inhibition of transcription and translation of the gene or production of that gene's protein. The inhibitory dose or "therapeutic dose" of the compound of the present invention may be determined by assessing the affects of the oligonucleotides on cell growth and tissue culture or tissue growth in an animal. A therapeutic dose in an amount sufficient to cause the required inhibitory effects. One skilled in the art will recognize the amount of oligonucleotide administered in a therapeutic dose is dependent upon the age, weight, kind of concurrent treatment and nature of the cell growth condition being treated. Those skilled in the art will know how to adjust the dose based on the circumstances. The amount of oligonucleotide in a therapeutic dose will include a sufficient amount to account for cellular uptake and binding. In the present invention, the percentage of guanosine necessary to preserve anti-viral activity is between 25 and 53% or more, or an amount sufficient to produce a three-dimensional structure stabilized by guanosine tetrads having at least two segments (runs) of at least two contiguous guanosines. The rest of the molecules are usually composed of thymidine, cytosine, xanthosine, adenine, uridine, 5-fluorodeoxy uridine, uracil or inosine nucleotides (ribo- or deoxyribo-) or their derivatives.

One skilled in the art readily recognizes that the 5' and 3' termini of the polynucleotide can have an attachment which may enhance stability and/or uptake into cells or cell nuclei. The backbone which connects the nucleotides can be a standard phosphodiester linkage or any modification of this linkage which may improve stability of the molecule and/or provide anti- cytokine and/or subsequent tumor growth inhibition activity of the molecule. Examples of a backbone linkage which is useful in the present invention in addition to the standard backbone is a phosphorothioate linkage. The present invention relates to a broad anti-cytokine spectrum of the oligonucleotides of the present invention and some of the mechanisms by which they exhibit their therapeutic properties. These compounds can form a triple helix according to the method of Hogan, et al., U.S. Serial No. 5,176,996, issued January 5, 1993, at selected sites in the TNF-α genes. This is observed because the oligonucleotides used in the present invention are designed as triple helix forming oligonucleotides which complex with portions of the human TNF-α gene. However, because these molecules can form inter- or intramolecular tetrads in physiologic conditions, very little material is available for TFO formation, therefore, TFO formation does not account for the total inhibition seen. A key aspect of the present invention is the presence of guanosine residues. Further, the oligonucleotides of the present invention are not designed as an antisense molecule. One embodiment of the present invention is a method of inhibiting cytokine production in cells comprising the step of administering a therapeutic dose of an oligonucleotide, said oligonucleotide having a composition of at least 70% guanosine and thymidine, being designed to form a colinear triplex with TNF-α gene and having at least 25% guanosine.

Although the oligonucleotide is designed to form a triplex with TNF-α gene, it does not necessarily have to form a triplex to function in the present invention.

In an alternate embodiment of the present invention, any of the procedures or methods described herein can have an oligonucleotide comprised of enough guanosine to contain at least two runs of at least two contiguous guanosines and to have a three-dimensional structure stabilized by guanosine tetrads.

The method of inhibiting cytokine production can also be used for the inhibition of TNF-α protein production.

Another alternative embodiment of the present invention is a method of inhibiting lipopolysaccharide (or other mitogens) induced TNF-α production in cells. In this procedure, a therapeutic dose of an oligonucleotide having a composition of guanosine sufficient to produce a three-dimensional structure stabilized by guanosine tetrads, form at least two runs of at least two contiguous guanosines, and designed to form a colinear triplex with the TNF-α gene, is delivered to the cells to be affected.

The method of inhibiting cytokine production and TNF-α protein production can also be used to inhibit tumor cells. This is especially appropriate in the inhibition of TNF-α responsive tumor cells, either autocrine or exocrine.

In an alternate embodiment of the present invention, any of the procedures or methods described herein can have an oligonucleotide that is at least 25% guanosine and at least 70% guanosine and thymidine. The following examples are offered by way of illustration and are not intended to limit the invention in any manner. EXAMPLE 1 Structural Forms

Structural forms of several different G-rich polynucleotides (oligonucleotides) useful in the present invention are shown below in combination with the sites of attachment as potential triplex-forming oligonucleotides. The oligonucleotides of the present invention have a hydrophobic moiety attached to the 3' terminus. The oligonucleotides shown below all have a polypropylamine (amine) or a cholesterol (chol. 2b) group attached to 3' terminus. The 3' capping group confers stability against nuclease digestion to these oligonucleotides. Their location is shown relative to mRNA start cite of the TNF-α gene.

TABLE I

J108 -58 to -33 relative to mRNA start site (7 bp from TATA);

5'- atgattctttccccgccctcctctcgccccaggg -3' duplex target 3*- tactaagaaaggggcgggaggagagcggggtccc -5'

J108-50 5'- ttgtttggggtgggtggtgtgtgggg -3' Amine

J108-57 5' -3" chol. 2b

J108-56 5' F F -3' chol. 2b

J108-58 5' 1 1 -3' chol. 2b

Where F=fluoro-deoxy uracil, I=imidazole and - connotes a base the same as the sequence above it.

TABLE II J109 -237 to -208; relative to the mRNA start site.

5'- atacccctcacactccccatcctccctgctccgat -3' duplex target

3'- tatggggagtgtgaggggtaggagggacgaggcta -5'

J109-50 5*- ggggtgtgtgtggggttggtgggttgtgg -3' 3' Amine J109-51 5' -3* 3' chol. 2b TABLE III J110 +1120 to +1153; 33 mer, 87.8% purine, 63.6% G

5*- ataccccctactttcacctccatccatcctcccccaag -3' duplex 3'- tatgggggatgaaagtggaggtaggtaggagggggttc -5'

J110-50 5'- gggggttgtttgtggtggttggttggtggggg -3' Amine

J110-51 5' -3' chol. 2b

TABLE IV Jill +1429 TO +1456; 27 mer, 100% purine, 66.6% G

5'- tgactctcctctccctctctccctccctccagc -3' duplex target 3'- actgagaggagagggagagagggagggaggtcg -5'

Jlll-50 5 - gtgtggtgtgggtgtgtgggtgggtgg -3' Amine

Jlll-51 5'- -3' chol. 26 additional oligonucleotides:

Jlll-01 5'- ggtgggtgggtgtgtgggtgtggtgtg -3' chol. 2b Jlll-56 5'- tgggtggggtgtgtgtgtgggtggggt -3' chol. 2b

EXAMPLE 2 LPS induced TNF-α production in THP-1 cells

THP-1 cells were cultured at 37° C in a 5% C0₂ atmosphere using RPMI media supplemented with 10% fetal bovine serum (FBS), 50 uM β- mercaptoethanol and antibiotics (penicillin and streptomycin). Cells were split 24 hours prior to use at 1 to 5 x 10⁵ cells/well in a 24 well culture dish. The cells are gently centrifuged down, washed once in medium without serum and resuspended in 0.5 ml of optimem (low serum medium, BRL) containing various concentrations of oligonucleotides. The cells were cultured thus for 3 hours at 37° C. After 3 hours the cells were again centrifuged, washed once in complete medium with FBS and resuspended in 0.5 ml of complete medium with the same concentration of oligonucleotide. The cells were then stimulated to produce TNF-α by the addition of 1.0, 0.25 or 0.1 ug/ml bacterial LPS. Cells were cultured at 37° C in the presence of LPS and samples of the medium collected at various time points. The presence of TNF-α in the culture medium was determined using a commercially available ELISA kit (R and D systems, or BMB).

EXAMPLE 3 U251 cell culture U251 (glioblastoma cell line) cells were plated in microtiter plate wells at 600 cells per well, and cultured in DMEM/Ham's F12 medium with 10% FBS. Oligonucleotide Jlll-51 was added at the indicated concentration on day 0. On days 0, 3, 5 and 7, post plating viable cells were assayed by formazan dye production from tetrazolium salts. Absorbance at 490 nm was used to determine the degree of dye metabolism. The degree of dye metabolism is used as an indicator of cell growth.

EXAMPLE 4 Inhibition by oligonucleotide

A. Inhibition of TNF-α Protein Production. As can be seen in Figure 1, Jlll-50 (3' amine modified oligonucleotide) was able to reduce the detectable levels of TNF-α in the culture medium 4 hours post-induction of TNF-α by bacterial LPS (Figure 1). The amount of LPS used to induce TNF- α expression in this experiment was 1 ug/ml. Little to no activity was observed when J108-50 was used in this experiment even up to 40 uM concentrations (Figure 1). However, when the level of LPS used to induce

TNF-α expression was reduced from 1 ug/ml to 0.1 ug/ml, then both Jlll-50 and J108-50 were able to significantly reduce TNF-α expression in these cells (Figure 2).

B. Tumor Inhibition. In the experiment designed to monitor the inhibition of the TNF-α responsive glioblastoma cell line (U251) it is apparent that concentrations of Jlll-51 (3'-cholesterol 2b modified version of Jlll-50) was able to significantly reduce the proliferation of these cells (in a non lethal fashion) at concentrations as low as 2 uM (Figure 3).

As can be seen from the data presented above, anti-TNF-α activity in tissue culture assays was observed for several G-Rich polynucleotides including Jlll-50 and J108-50. In addition, the G-Rich polynucleotide, Jlll-51 was able to inhibit the proliferation of U251 cells in culture. Thus, oligonucleotides containing a high percentage of guanosine residues make potent anti-cytokine agents and are useful as anti-tumor agents for those tumors which are regulated by cytokines. All patents and publications mentioned in this specification are indicative of the levels of those skilled in the art to which the invention pertains. All patents and publications are herein incorporated by reference to the same extent as if each individual publication was specifically and individually indicated to be incorporated by reference. One skilled in the art will readily appreciate that the present invention is well adapted to carry out the objects and obtain the ends and advantages mentioned, as well as those inherent therein. The oligonucleotides, compounds, methods, procedures and techniques described herein are presently representative of the preferred embodiments, are intended to be exemplary and are not intended as limitations on the scope. Changes therein and other uses will occur to those skilled in the art which are encompassed within the spirit of the invention and are defined by the scope of the appended claims.

SEQUENCE LISTING

(1) GENERAL INFORMATION:

(i) APPLICANT: Rando, Robert F. Santon, June

(ii) TITLE OF INVENTION: The Inhibition of Cytokine Production by

Oligonucleotides and Inhibition of Tumors Which are Responsive to Cytokine Factors

(iii) NUMBER OF SEQUENCES: 10

(iv) CORRESPONDENCE ADDRESS:

(A) ADDRESSEE: Fulbright & Jaworski

(B) STREET: 1301 McKinne , Suite 5100

(C) CITY: Houston

(D) STATE: Texas (E) COUNTRY: US

(F) ZIP: 77010-3095

(v) COMPUTER READABLE FORM:

(A) MEDIUM TYPE: Floppy disk

(B) COMPUTER: IBM PC compatible (C) OPERATING SYSTEM: PC-DOS/MS-DOS

(D) SOFTWARE: Patentln Release #1.0, Version #1.25

(vi) CURRENT APPLICATION DATA:

(A) APPLICATION NUMBER:

(B) FILING DATE: (C) CLASSIFICATION: (viii) ATTORNEY/AGENT INFORMATION:

(A) NAME: Paul, Thomas D.

(B) REGISTRATION NUMBER: 32,714

(C) REFERENCE/DOCKET NUMBER: D-5669

(ix) TELECOMMUNICATION INFORMATION:

(A) TELEPHONE: 713/651-5151

(B) TELEFAX: 713/651-5246

(C) TELEX: 762829

(2) INFORMATION FOR SEQ ID NO:l:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 34 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: DNA (genomic)

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:l:

ATGATTCTTT CCCCGCCCTC CTCTCGCCCC AGGG 34

(2) INFORMATION FOR SEQ ID NO:2:

(i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 26 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single (D) TOPOLOGY: linear

(ii) MOLECULE TYPE: DNA (genomic)

(ix) FEATURE:

(A) NAME/KEY: misc_feature

(B) LOCATION: 1

(D) OTHER INFORMATION: /note= "n = thymidine, fluoro-deoxy uracil or imidazole"

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:

TTGTTTGGGG NGGGTGGTGT GNGGGG 26

(2) INFORMATION FOR SEQ ID NO:3:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 35 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single (D) TOPOLOGY: linear

(ii) MOLECULE TYPE: DNA (genomic)

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:

ATACCCCTCA CACTCCCCAT CCTCCCTGCT CCGAT 35

(2) INFORMATION FOR SEQ ID NO:4: (i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 29 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: DNA (genomic)

(xi) SEQUENCEDESCRIPTION: SEQ ID NO:4:

GGGGTGTGTG TGGGGTTGGT GGGTTGTGG 29

(2) INFORMATION FORSEQ ID NO:5:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 38 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: DNA (genomic)

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:5:

ATACCCCCTA CTTTCACCTC CATCCATCCT CCCCCAAG 38

(2) INFORMATION FOR SEQ ID NO:6:

(i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 32 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: DNA (genomic)

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:6:

GGGGGTTGTT TGTGGTGGTT GGTTGGTGGG GG 32

(2) INFORMATION FOR SEQ ID NO:7:

(i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 33 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: DNA (genomic)

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:7:

TGACTCTCCT CTCCCTCTCT CCCTCCCTCC AGC 33

(2) INFORMATION FOR SEQ ID NO:8:

(i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 27 base pairs (B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: DNA (genomic)

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:8:

GTGTGGTGTG GGTGTGTGGG TGGGTGG 27

(2) INFORMATION FOR SEQ ID NO:9:

(i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 27 base pairs (B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: DNA (genomic)

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:9:

GGTGGGTGGG TGTGTGGGTG TGGTGTG 27

(2) INFORMATION FOR SEQ ID NO:10:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 27 base pairs

(B) TYPE: nucleic acid (C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: DNA (genomic)

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:10:

TGGGTGGGGT GTGTGTGTGG GTGGGGT 27

Claims

What is claimed is:CLAIMS

1. A method for inhibiting cytokine production in cells comprising the step of administering a therapeutic dose of an oligonucleotide, said oligonucleotide having a composition of guanosine sufficient to produce a three-dimensional structure stabilized by guanosine tetrads or having a composition of at least two runs of at least two contiguous guanosines.

2. The method of claim 1, wherein said oligonucleotide forms inter- or intramolecular tetrads.

3. The method of claim 1, wherein said oligonucleotide has a composition of at least 70% guanosine and thymidine or their naturally- occurring or synthetic derivatives and wherein at least 25% is guanosine or guanosine derivatives.

4. A method for inhibiting TNF-α protein production in cells, comprising the step of administering a therapeutic dose of an oligonucleotide, said oligonucleotide having a composition of guanosine sufficient to produce a three-dimensional structure stabilized by guanosine tetrads or having a composition of at least two runs of at least two contiguous guanosines.

5. The method of claim 4, wherein said oligonucleotide forms inter- or intramolecular tetrads.

6. The method of claim 4, wherein said oligonucleotide has a composition of at least 70% guanosine and thymidine or their naturally- occurring or synthetic derivatives and wherein at least 25% is guanosine or guanosine derivatives.

7. A method for inhibiting lipopolysaccharide or other mitogen- induced TNF-α protein production in cells, comprising the step of administering a therapeutic dose of an oligonucleotide, said oligonucleotide having a composition of guanosine sufficient to produce a three-dimensional structure stabilized by guanosine tetrads or having at least two runs of at least two contiguous guanosines, and designed to form a colinear triplex with a TNF-α gene.

8. The method of claim 7, wherein said oligonucleotide forms inter- or intramolecular tetrads.

9. The method of claim 7, wherein said oligonucleotide has a composition of at least 70% guanosine and thymidine or their naturally- occurring or synthetic derivatives and wherein at least 25% is guanosine or guanosine derivatives.

10. A method for inhibiting the growth of TNF-α responsive cells, comprising the step of administering a therapeutic dose of an oligonucleotide, said oligonucleotide having a composition of guanosine sufficient to produce a three-dimensional structure stabilized by guanosine tetrads or having a composition of at least two runs of at least two contiguous guanosines.

11. The method of claim 10, wherein said oligonucleotide forms inter- or intramolecular tetrads.

12. The method of claim 10, wherein said oligonucleotide has a composition of at least 70% guanosine and thymidine or their naturally- occurring or synthetic derivatives and wherein at least 25% is guanosine or guanosine derivatives.

13. The method of claim 10 wherein the cells are glioblastoma cells.

14. The method of claim 1, 4, 7 or 10, wherein the oligonucleotide is selected from the group of sequences consisting of SEQ. ID. No. 2, SEQ. ID. No. 4, SEQ. ID. No. 6, SEQ. ID. No. 8, SEQ. ID. No. 9, and SEQ. ID. No. 10; and wherein N in SEQ. ID. No. 2 isselected from the group consisting of thymine, fluoro-deoxy uracil, and imidazole.

15. The method of claim 14, wherein a propanol amine group is attached to the 3' terminus of the oligonucleotide.

16. The method of claim 14, where a hydrophobic moiety is linked to the 3' terminus of the oligonucleotide.

17. The method of claim 16 where the hydrophobic moiety is cholesterol.

18. An oligonucleotide selected from the group of sequences consisting of SEQ. ID. No. 2, SEQ. ID. No. 4, SEQ. ID. No. 6, SEQ. ID. No.

8, SEQ. ID. No. 9, and SEQ. ID. No. 10.; and wherein N in SEQ. ID. No. 2 is selected from the group consisting of thymine, fluoro-deoxy uracil, and imidazole

19. The oligonucleotides of claim 18, wherein a propanol amine group is attached to the 3' terminus of the oligonucleotide.

20. The oligonucleotide of claim 19, wherein a hydrophobic moiety is linked to the 3' terminus of the oligonucleotide.

21. The oligonucleotide of claim 20, wherein the hydrophobic moiety is cholesterol.