Antiviral oligonucleotides having a conserved G4 core sequence

Abstract

Modified oligonucleotides having a conserved G 4 sequence and a sufficient number of flanking nucleotides to significantly inhibit the activity of a virus or phospholipase A 2 or to modulate the telomere length of a chromosome are provided. G 4 quartet oligonucleotide structures are also provided. Methods of prophylaxis, diagnostics and therapeutics for viral-associated diseases and diseases associated with elevated levels of phospholipase A 2 are also provided. Methods of modulating telomere length of a chromosome are also provided; modulation of telomere length is believed to play a role in the aging process of a cell and in control of malignant cell growth.

1 . A chemically modified oligonucleotide having no more than about 27 nucleic acid base units, said oligonucleotide comprising at least one GGGG sequence or at least two GGG sequences and a sufficient number of flanking nucleotides to significantly inhibit the activity of a virus or phospholipase A 2  or to modulate the telomere length of a chromosome.  
     
     
         2 . An oligonucleotide of  claim 1  wherein significant inhibition of viral or enzyme activity is at least 50% inhibition.  
     
     
         3 . An oligonucleotide of  claim 1  wherein the virus is HIV, HSV, HCMV or influenza virus.  
     
     
         4 . An oligonucleotide of  claim 3  wherein the virus is HSV.  
     
     
         5 . An oligonucleotide of  claim 4  wherein the oligonucleotide is selected from the group consisting of: SEQ ID NO: 21, SEQ ID NO: 1, SEQ ID NO: 8, SEQ ID NO: 12, SEQ ID NO: 16, SEQ ID NO: 22, SEQ ID NO: 28, SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO: 33, SEQ ID NO: 35, SEQ ID NO: 36, SEQ ID NO: 37, SEQ ID NO: 38, SEQ ID NO: 39, SEQ ID NO: 42, SEQ ID NO: 47, SEQ ID NO: 48 and SEQ ID NO: 50.  
     
     
         6 . An oligonucleotide of  claim 4  having a sequence shown in Table 8.  
     
     
         7 . An oligonucleotide of  claim 6  having a sequence selected from the group consisting of SEQ ID NO: 124, SEQ ID NO:126, SEQ ID NO: 127, SEQ ID NO: 128, SEQ ID NO: 129, SEQ ID NO: 130 and SEQ ID NO: 133.  
     
     
         8 . An oligonucleotide of  claim 1  having the sequence (N X G 4 N Y ) Q  wherein X and Y are independently 1 to 8 and Q is 1 to 4.  
     
     
         9 . An oligonucleotide of  claim 8  having the sequence NNGGGGNN.  
     
     
         10 . An oligonucleotide of  claim 9  which has at least one phosphorothioate intersugar linkage and which has the sequence GNGGGGTN.  
     
     
         11 . An oligonucleotide of  claim 1  having the sequence (G 4 N X G 4 ) Q  wherein X is 1 to 8 and Q is 1 to 3.  
     
     
         12 . An oligonucleotide of  claim 1  having the sequence (N X G 3-4 ) Q N X  wherein X is 1 to 8 and Q is 1 to 6.  
     
     
         13 . An oligonucleotide of  claim 1  which has at least one phosphorothioate intersugar (backbone) linkage.  
     
     
         14 . An oligonucleotide of  claim 1  wherein each of the nucleosides is in the alpha (α) anomeric configuration.  
     
     
         15 . An oligonucleotide of  claim 1  which is a chimeric oligonucleotide.  
     
     
         16 . A phosphorothioate oligonucleotide having SEQ ID NO: 21.  
     
     
         17 . A phosphorothioate oligonucleotide having the sequence TTGGGGTT.  
     
     
         18 . The oligonucleotide of  claim 17  wherein each of the nucleotides of the oligonucleotide is in the alpha (α) anomeric configuration.  
     
     
         19 . A method for inhibiting the activity of a virus comprising contacting the virus with a chemically modified oligonucleotide having no more than 27 nucleic acid base units comprising at least one GGGG sequence and at least two GGG sequences and a sufficient number of flanking nucleotides to significantly inhibit the activity of the virus.  
     
     
         20 . The method of  claim 19  wherein significant inhibition of viral activity is at least 50% inhibition.  
     
     
         21 . The method of  claim 19  wherein the virus is HIV, HSV, HCMV or influenza virus.  
     
     
         22 . The method of  claim 21  wherein the virus is HSV.  
     
     
         23 . The method of  claim 22  wherein the oligonucleotide is selected from the group consisting of: SEQ ID NO: 21, SEQ ID NO: 1, SEQ ID NO: 8, SEQ ID NO: 12, SEQ ID NO: 16, SEQ ID NO: 22, SEQ ID NO: 28, SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO: 33, SEQ ID NO: 35, SEQ ID NO: 36, SEQ ID NO: 37, SEQ ID NO: 38, SEQ ID NO: 39, SEQ ID NO: 42, SEQ ID NO: 47, SEQ ID NO: 48 and SEQ ID NO: 50.  
     
     
         24 . The method of  claim 22  wherein the oligonucleotide has a sequence shown in Table 8.  
     
     
         25 . The method of  claim 24  wherein the oligonucleotide has a sequence selected from the group consisting of SEQ ID NO: 124, SEQ ID NO: 126, SEQ ID NO: 127, SEQ ID NO: 128, SEQ ID NO: 129, SEQ ID NO: 130 and SEQ ID NO: 133.  
     
     
         26 . The method of  claim 19  wherein said oligonucleotide has the sequence (N X G 4 N Y ) Q  wherein X and Y are independently 1 to 8 and Q is 1 to 4.  
     
     
         27 . The method of  claim 26  wherein said oligonucleotide has the sequence NNGGGGNN.  
     
     
         28 . The method of  claim 27  wherein the oligonucleotide has at least one phosphorothioate intersugar linkage and the sequence GNGGGGTN.  
     
     
         29 . The method of  claim 19  wherein said oligonucleotide has the sequence (G 4 N X G 4 ) Q  wherein X is 1 to 8 and Q is 1 to 3.  
     
     
         30 . The method of  claim 19  wherein said oligonucleotide has the sequence (N X G 3-4 ) Q N X  wherein X is 1 to 8 and Q is 1 to 6.  
     
     
         31 . The method of  claim 19  wherein said oligonucleotide comprises a sequence identified in Table 1, Table 2 or Table 3.  
     
     
         32 . The method of  claim 19  wherein said oligonucleotide has at least one phosphorothioate intersugar (backbone) linkage.  
     
     
         33 . The method of  claim 19  wherein each of the nucleosides of the oligonucleotide is in the alpha (α) anomeric configuration.  
     
     
         34 . The method of  claim 19  wherein the oligonucleotide is a chimeric oligonucleotide.  
     
     
         35 . A method for inhibiting the activity of a virus comprising contacting the virus with a phosphorothioate oligonucleotide having SEQ ID NO: 21.  
     
     
         36 . A method for inhibiting the activity of a virus comprising contacting the virus with a phosphorothioate oligonucleotide having the sequence TTGGGGTT.  
     
     
         37 . The method of  claim 36  wherein each of the nucleotides of the oligonucleotide is in the alpha (α) anomeric configuration.  
     
     
         38 . The method of  claim 36  wherein the virus is HIV.  
     
     
         39 . A method for inhibiting phospholipase A 2  enzyme activity comprising contacting a cell with a chemically modified oligonucleotide having no more than about 27 nucleic acid base units comprising at least one GGGG sequence or at least two GGG sequences and a sufficient number of flanking nucleotides to significantly inhibit the activity of phospholipase A 2 .  
     
     
         40 . The method of  claim 39  wherein the phospholipase A 2  enzyme activity is inhibited by greater than 50%.  
     
     
         41 . The method of  claim 39  wherein said oligonucleotide comprises a sequence selected from the group consisting of: SEQ ID NO:12, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:26, SEQ ID NO:42, SEQ ID NO:47, SEQ ID NO:50, SEQ ID NO:65, SEQ ID NO:66, SEQ ID NO:68, SEQ ID NO:71, SEQ ID NO:73, SEQ ID NO:83, SEQ ID NO:97, SEQ ID NO:98, SEQ ID NO:99, SEQ ID NO:104, SEQ ID NO:105, SEQ ID NO:106, SEQ ID NO:107 and TGGGG.  
     
     
         42 . The method of  claim 39  wherein said oligonucleotide has at least one phosphorothioate intersugar (backbone) linkage.  
     
     
         43 . A method of treating a viral-associated disease comprising administering to an animal having a viral-associated disease a therapeutically effective amount of a chemically modified oligonucleotide having no more than about 27 nucleic acid base units comprising at least one GGGG sequence and at least two GGG sequences and a sufficient number of flanking nucleotides to significantly inhibit the activity of the virus.  
     
     
         44 . The method of  claim 43  wherein significant inhibition of viral activity is at least 50% inhibition.  
     
     
         45 . The method of  claim 43  wherein the virus is HIV, HSV, HCMV or influenza virus.  
     
     
         46 . The method of  claim 45  wherein the virus is HSV.  
     
     
         47 . The method of  claim 46  wherein the oligonucleotide is selected from the group consisting of: SEQ ID NO: 21, SEQ ID NO: 1, SEQ ID NO: 8, SEQ ID NO: 12, SEQ ID NO: 16, SEQ ID NO: 22, SEQ ID NO: 28, SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO: 33, SEQ ID NO: 35, SEQ ID NO: 36, SEQ ID NO: 37, SEQ ID NO: 38, SEQ ID NO: 39, SEQ ID NO: 42, SEQ ID NO: 47, SEQ ID NO: 48 and SEQ ID NO: 50.  
     
     
         48 . The method of  claim 46  wherein the nucleotide has a sequence shown in Table 8.  
     
     
         49 . The method of  claim 48  wherein the oligonucleotide has a sequence selected from the group consisting of SEQ ID NO: 124, SEQ ID NO: 126, SEQ ID NO: 127, SEQ ID NO: 128, SEQ ID NO: 129, SEQ ID NO: 130 and SEQ ID NO: 133.  
     
     
         50 . The method of  claim 43  wherein said oligonucleotide has the sequence (N X G 4 N Y ) Q  wherein X and Y are independently 1 to 8 and Q is 1 to 4.  
     
     
         51 . The method of  claim 50  wherein said oligonucleotide has the sequence NNGGGGNN.  
     
     
         52 . The method of  claim 51  wherein said oligonucleotide has at least one phosphorothioate intersugar linkage and the sequence GNGGGGTN.  
     
     
         53 . The method of  claim 43  wherein said oligonucleotide has the sequence (G 4 N X G 4 ) Q  wherein X is 1 to 8 and Q is 1 to 3.  
     
     
         54 . The method of  claim 43  wherein said oligonucleotide has the sequence (N X G 3-4 ) Q N X  wherein X is 1 to 8 and Q is 1 to 6.  
     
     
         55 . The method of  claim 43  wherein said oligonucleotide comprises a sequence identified in Table 1, Table 2 or Table 3.  
     
     
         56 . The method of  claim 43  wherein said oligonucleotide has at least one phosphorothioate intersugar (backbone) linkage.  
     
     
         57 . The method of  claim 43  wherein each of the nucleotides of the oligonucleotide is in the alpha (α) anomeric configuration.  
     
     
         58 . The method of  claim 43  wherein the oligonucleotide is a chimeric oligonucleotide.  
     
     
         59 . A method of treating a viral-associated disease comprising contacting the virus with a phosphorothioate oligonucleotide having SEQ ID NO: 21.  
     
     
         60 . A method of treating a viral-associated disease comprising contacting the virus with a phosphorothioate oligonucleotide having the sequence TTGGGGTT.  
     
     
         61 . The method of  claim 60  wherein each of the nucleotides of the oligonucleotide is in the alpha (α) anomeric configuration.  
     
     
         62 . The method of  claim 60  wherein the virus is HIV.  
     
     
         63 . A method of treating an inflammatory disease or a neurological disorder associated with phospholipase A 2  enzyme activity comprising administering to an animal having such an inflammatory disease or neurological disease a therapeutically effective amount of a chemically modified oligonucleotide having no more than about 27 nucleic acid base units comprising at least one GGGG sequence and at least two GGG sequences and a sufficient number of flanking nucleotides to significantly inhibit the activity of phospholipase A 2 .  
     
     
         64 . The method of  claim 63  wherein significant inhibition of enzyme activity is at least 50% inhibition.  
     
     
         65 . The method of  claim 63  wherein said oligonucleotide comprises a sequence selected from the group consisting of: SEQ ID NO:12, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:26, SEQ ID NO:42, SEQ ID NO:47, SEQ ID NO:50, SEQ ID NO:65, SEQ ID NO:66, SEQ ID NO:68, SEQ ID NO:71, SEQ ID NO:73, SEQ ID NO:83, SEQ ID NO:97, SEQ ID NO:98, SEQ ID NO:99, SEQ ID NO: 104, SEQ ID NO: 105, SEQ ID NO:106, SEQ ID NO:107 and TGGGG.  
     
     
         66 . A method of modulating telomere length of a chromosome comprising contacting a chromosome with a chemically modified oligonucleotide 6 to 27 nucleic acid base units in length having the sequence (N X G 3-4 ) Q N X  wherein X is 1-8 and Q is 1-5.  
     
     
         67 . A method for inhibiting the division of a malignant cell comprising contacting a malignant cell with a chemically modified oligonucleotide having 6 to 27 nucleic acid base units and having the sequence (N X G 3-4 ) Q N X  wherein X is 1-8 and Q is 1-5.  
     
     
         68 . A compound comprising a G-quartet structure of phosphorothioate oligonucleotides each oligonucleotide having the sequence TxG4Ty where x and y are independently 0 to 8.  
     
     
         69 . The compound of  claim 68  wherein the nucleotides of at least one of the oligonucleotides of the G-quartet structure are in the alpha (α) anomeric configuration.  
     
     
         70 . The compound of  claim 68  wherein x is 2 and y is 2.  
     
     
         71 . The compound of  claim 68  wherein x is 0 and y is 2.  
     
     
         72 . The compound of  claim 68  wherein x is 3 and y is 3.  
     
     
         73 . The compound of  claim 68  wherein each oligonucleotide has the sequence (TxG4Ty)q where x and y are independently 0 to 8 and q is from 1 to 10.  
     
     
         74 . A method for inhibiting the activity of human immunodeficiency virus comprising administering to a cell infected with said virus a compound comprising a G-quartet structure of phosphorothioate oligonucleotides each oligonucleotide having the sequence TxG4Ty where x and y are independently 0 to 8 in an amount sufficient to inhibit the activity of the virus.  
     
     
         75 . The method of  claim 74  wherein inhibition of viral activity is at least 50% inhibition.  
     
     
         76 . The method of  claim 74  wherein a compound in which x is 2 and y is 2 is administered to a cell infected with human immunodeficiency virus.  
     
     
         77 . The method of  claim 75  wherein a compound in which x is 0 and y is 2 is administered to a cell infected with human immunodeficiency virus.  
     
     
         78 . The method of  claim 75  wherein a compound in which x is 3 and y is 3 is administered to a cell infected with human immunodeficiency virus.  
     
     
         79 . A method for treating a patient infected with human immunodeficiency virus comprising administering to said patient a compound comprising a G-quartet structure of phosphorothioate oligonucleotides having the sequence TxG4Ty where x and y are independently 0 to 8 in an amount sufficient to inhibit the activity of the virus.  
     
     
         80 . The method of  claim 79  wherein a compound in which x is 2 and y is 2 is administered to said patient infected with human immunodeficiency virus.  
     
     
         81 . The method of  claim 79  wherein a compound in which x is 0 and y is 2 is administered to said patient infected with human immunodeficiency virus.  
     
     
         82 . The method of  claim 79  wherein a compound in which x is 3 and y is 3 is administered to said patient infected with human immunodeficiency virus.  
     
     
         83 . A pharmaceutical composition comprising a compound comprising a G-quartet structure of phosphorothioate oligonucleotides having the sequence TxG4Ty where x and y are independently 0 to 8 and a pharmaceutically acceptable carrier.  
     
     
         84 . A prophylactic device coated with a compound comprising a G-quartet structure of phosphorothioate oligonucleotides having the sequence TxG4Ty where x and y are independently 0 to 8.