Modulation of telomere length by oligonucleotides having a G-core sequence

• US 7,067,497 B2
• Filed: 01/02/2002
• Issued: 06/27/2006
• Est. Priority Date: 09/29/1992
• Status: Expired due to Fees

• Current Assignee: ISIS Pharmaceuticals Incorporated
• Original Assignee: ISIS Pharmaceuticals Incorporated
• Inventors: Bennett, C. Frank, Ecker, David J., Vickers, Timothy A., Hanecak, Ronnie C., Brown-Driver, Vickie L., Anderson, Kevin P., Wyatt, Jacqueline R., Chiang, Ming-Yi
• Primary Examiner(s): Guzo, David
• Application Number:
  US10/038,335
• Publication Number:
  US 20030096776A1
• Time in Patent Office: 1,637 Days
• Field of Search: 536/23.1, 536/24.5, 536/25.2, 514/44
• US Class Current: 514/44.00A
• CPC Class Codes: A61K 31/70 Carbohydrates; Sugars; Derivatives thereof sorbitol A61K31/047
• CPC Class Codes: A61K 38/00 Medicinal preparations containing peptides peptides containing beta-lactam rings A61K31/00; cyclic dipeptides not having in their molecule any other peptide link than those which form their ring, e.g. piperazine-2,5-diones, A61K31/00; ergot alkaloids of the cyclic peptide type A61K31/48; containing macromolecular compounds having statistically distributed amino acid units A61K31/74; medicinal preparations containing antigens or antibodies A61K39/00; medicinal preparations characterised by the non-active ingredients, e.g. peptides as drug carriers, A61K47/00
• CPC Class Codes: C07H 21/00 Compounds containing two or more mononucleotide units having separate phosphate or polyphosphate groups linked by saccharide radicals of nucleoside groups, e.g. nucleic acids
• CPC Class Codes: C12N 15/1133 against herpetoviridae, e.g. HSV
• CPC Class Codes: C12N 15/1137 against enzymes viral enzymes C12N15/1131; receptors C12N15/1138
• CPC Class Codes: C12N 15/117 Nucleic acids having immunomodulatory properties, e.g. containing CpG-motifs
• CPC Class Codes: C12N 2310/151 more than 3 strands, e.g. tetrads, H-DNA
• CPC Class Codes: C12N 2310/18 acting by a non-sequence specific mechanism other than C12N2310/16 or C12N2310/17
• CPC Class Codes: C12N 2310/315 Phosphorothioates
• CPC Class Codes: C12N 2310/321 2'-O-R Modification
• CPC Class Codes: C12N 2310/346 having a combination of backbone and sugar modifications
• CPC Class Codes: C12N 2310/3521 Methyl
• CPC Class Codes: C12Q 1/701 Specific hybridization probes
• CPC Class Codes: C12Y 207/07049 RNA-directed DNA polymerase (2.7.7.49), i.e. telomerase or reverse-transcriptase
• CPC Class Codes: C12Y 301/01004 Phospholipase A2 (3.1.1.4)

Abstract

Modified oligonucleotides having a GGG motif sequence and a sufficient number of flanking nucleotides to modulate the telomere length of a chromosome are provided. Methods of modulating telomere length of a mammalian chromosome in vitro and in vivo are also provided, as are methods for inhibiting the division of a malignant mammalian cell and for modulating the effects of cellular aging.

Claims

1. A method for inhibiting the division of a malignant mammalian cell comprising contacting said malignant mammalian cell with a chemically modified oligonucleotide having no more than about 27 nucleic acid base units, said oligonucleotide having the sequence (N_xG_3-4)_QN_X wherein X is 1 to 8 and Q is 1 to 6, wherein said oligonucleotide modulates mammalian telomere length.

2. The method of claim 1 which is carried out in vitro.

3. The method of claim 1 which is carried out in vivo.

4. A chemically modified oligonucleotide having no more than about 27 nucleic acid base units, said oligonueleotide having the sequence (N_xG₄)_QN_X wherein X is 1 to 8 and Q is 1 to 6.

5. The oligonucleotide of claim 4 which has at least one phosphorothioute linkage.

6. The oligonucleotide of claim 4 which has at least one 2′

modification of a sugar of said oligonucleotide.

7. The oligonucleotide of claim 4 which is a chimeric oligonucleotide.

8. The otigonucleotide of claim 4 wherein said chemical modification is:

a backbone modification selected from the group consisting of chiral phosphorothioate, phosphorodithioate, phosphotriester, aminoalkylphosphotriester, methyl phosphonate, 3′

-alkylene phosphonate, 5′

-alkylene phosphonate, chiral phosphonate, phosphinate, phosphoramidate, thionoalkyiphosphonate, thionoalkylphosphotriester, selenophosphate, boranophosphate, morpholino, siloxane, sulfide, sulfoxide, sulfone, formacetyl, thioformacetyl, methylene formacetyl, thioformacetyl, riboacetyl, alkene containing backbone, sulfamate, methyleneimino, methylenehydrazino, sulfonate, sulfonamide, and amide;

or

a modified 2′

sugar moiety selected from the group consisting of F, N-alkyl, O-alkenyl, S-alkenyl, N-alkenyl, O-alkynyl, S-alkynyl, N-alkynyl, O-alkyl-O-alkyl, C₁ to C₁₀ lower alkyl, substituted lower alkyl, alkenyl, alkynyl, alkaryl, aralkyl, O-alkaryl, O-aralkyl, SH, SCH₃, OCN, Cl, Br, CN, CF₃, OCF₃, SOCH_{3, SO2}CH, ONO₂, NO₂, N₃, NH₂, heterocycloalkyl, heterocycloalkaryl, aminoalkylamino, polyalkylamino, and substituted silyl, wherein the alkyl, alkenyl and alkynyl may be substituted or unsubstituted C₁ to C₁₀ alkyl or C₂ to C₁₀ alkenyl and alkynyl;

or

a modified nucleobases selected from the group consisting of 5-methylcytosine, 5-hydroxymethyl cytosine, xanthine, hypoxanthine, 2-aminoadenine, 6-methyl adenine, 6-methyl guanine, 2-propyl adenine, 2-propyl guanine, 2-thiouracil, 2-thiothymine, 2-thiocytosine, 5-halouracil, 5-holocytosine, 5-propynyl uracil, 5-propynyl cytosine, 6azo uracil, 6-azo cytosine, 6-azo thymine, 5-uracil, 4-thiouracil, 8-halo guanine, 8-amino guanine, 8-thiol guanine, 8-thioalkyl guanine, 8-hydroxyl guanine, 8-halo adenine, 8-amino adenine, 8-thiol adenine, 8-thioalkyl adenine, 8-hydroxyl adenine, 5-halo uracil, 5-bromo uracil, 5-trifluoromethyl uracil, 5-halo cytosine, 5-bromo cytosine, 5-trifluoromethyl cytosine, 7-methylguanine, 7-methyladenine, 2-F-adenine, 2-amino-adenine, 8-azaguanine, 8-azaadenine, 7-deazaguanine, 7-deazaudenine, 3-deazaguanine, 3-deazeadenine, phenoxazine cytidine(1H-pyrimido(5,4-b)(1,4)benzoxazin-2(3H)-one), phenothiazine cytidine (1H-pyrimido(5,4-b)(1,4)benzothiazin-2(3H)-one), substituted phenoxazine cytidine (e.g. 9-(2-aminoethoxy)-H-pyrimido(5,4-b)(1,4)benzoxazin-2(3H)-one), carbazole cytidine (2H-pyrimido(4,5-b)indol-2-one), pyridoindole cytidine (H-pyrido(3′

,2′

;

4,5)pyrrolo(2,3-d)pyrimidin-2-one), 7-deaza-adenine, 7-deazaguanosine, 2-amninopyridine, and 2-pyridone.

9. The oligonucleotide of claim 4 wherein said oligonucleotide modulates mammalian telomere length.

10. The oligonucleotide of claim 8 wherein the 2′

sugar modification is —

O—

CH₂CH₂OCH₃, —

O(CH₂ON(CH₃)₂, —

O—

CH₂—

O—

CH₂—

N(CH₂)₂, —

O—

CH₃, —

OCH₂CH₂CH₂NH₂, —

CH₂CH═

CH₂), —

F, —

O((CH₂)_nO)_mCH₃, —

O(CH₂)_nOCH₃, —

O(CH)₂)_n NH₂, —

O(CH₂)_nCH₃, —

O(CH₂)_nONH₂, or —

O(CH₂)_nON((CH₂)_nCH₃))₂, where n and m are from 1 to about 10.

15. The otigonucleotide of claim 4 wherein said chemical modification is:

a backbone modification selected from the group consisting of chiral phosphorothioate, phosphorodithioate, phosphotriester, aminoalkylphosphotriester, methyl phosphonate, 3′

-alkylene phosphonate, 5′

-alkylene phosphonate, chiral phosphonate, phosphinate, phosphoramidate, thionoalkyiphosphonate, thionoalkylphosphotriester, selenophosphate, boranophosphate, morpholino, siloxane, sulfide, sulfoxide, sulfone, formacetyl, thioformacetyl, methylene formacetyl, thioformacetyl, riboacetyl, alkene containing backbone, sulfamate, methyleneimino, methylenehydrazino, sulfonate, sulfonamide, and amide;

or

a modified 2′

sugar moiety selected from the group consisting of F, N-alkyl, O-alkenyl, S-alkenyl, N-alkenyl, O-alkynyl, S-alkynyl, N-alkynyl, O-alkyl-O-alkyl, C₁ to C₁₀ lower alkyl, substituted lower alkyl, alkenyl, alkynyl, alkaryl, aralkyl, O-alkaryl, O-aralkyl, SH, SCH₃, OCN, Cl, Br, CN, CF₃, OCF₃, SOCH₃, SO₂CH, ONO₂, NO₂, N₃, NH₂, heterocycloalkyl, heterocycloalkaryl, aminoalkylamino, polyalkylamino, and substituted silyl, wherein the alkyl, alkenyl and alkynyl may be substituted or unsubstituted C₁ to C₁₀ alkyl or C₂ to C₁₀ alkenyl and alkynyl;

or

a modified nucleobases selected from the group consisting of 5-methylcytosine, 5-hydroxymethyl cytosine, xanthine, hypoxanthine, 2-aminoadenine, 6-methyl adenine, 6-methyl guanine, 2-propyl adenine, 2-propyl guanine, 2-thiouracil, 2-thiothymine, 2-thiocytosine, 5-halouracil, 5-holocytosine, 5-propynyl uracil, 5-propynyl cytosine, 6azo uracil, 6-azo cytosine, 6-azo thymine, 5-uracil, 4-thiouracil, 8-halo guanine, 8-amino guanine, 8-thiol guanine, 8-thioalkyl guanine, 8-hydroxyl guanine, 8-halo adenine, 8-amino adenine, 8-thiol adenine, 8-thioalkyl adenine, 8-hydroxyl adenine, 5-halo uracil, 5-bromo uracil, 5-trifluoromethyl uracil, 5-halo cytosine, 5-bromo cytosine, 5-trifluoromethyl cytosine, 7-methylguanine, 7-methyladenine, 2-F-adenine, 2-amino-adenine, 8-azaguanine, 8-azaadenine, 7-deazaguanine, 7-deazaudenine, 3-deazaguanine, 3-deazeadenine, phenoxazine cytidine(1H-pyrimido(5,4-b)(1,4)benzoxazin-2(3H)-one), phenothiazine cytidine (1H-pyrimido(5,4-b)(1,4)benzothiazin-2(3H)-one), substituted phenoxazine cytidine (e.g. 9-(2-aminoethoxy)-H-pyrimido(5,4-b)(1,4)benzoxazin-2(3H)-one), carbazole cytidine (2H-pyrimido(4,5-b)indol-2-one), pyridoindole cytidine (H-pyrido(3′

,2′

;

4,5)pyrrolo(2,3-d)pyrimidin-2-one), 7-deaza-adenine, 7-deazaguanosine, 2-amninopyridine, and 2-pyridone.

16. The oligonucleotide of claim 15 wherein the 2′

sugar modification is —

O—

CH₂CH₂OCH₃, —

O(CH₂)₂ON(CH₃)₂, —

O—

CH₂—

O—

CH₂—

N(CH₂)₂, —

O—

CH₃, —

OCH₂CH₂CH₂NH₂, —

CH₂—

CH═

CH₂), —

F, —

O((CH₂)_nO)_mCH₃, —

O(CH₂)_nOCH₃, —

O(CH₂)_nNH₂, —

O(CH₂)_nCH₃, —

O(CH₂)_nONH₂, or —

O(CH₂)_nON((CH₂)_nCH₃))₂, where n and m are from 1 to about 10.

11. A chemically modified oligonucleotide having no more than about 27 nucleic acid base units, said oligonucleotide having the sequence (N_xG₄N_y)_Q or (G₄N_xG₄)_Q wherein X is 1 to 8, Y is 1 to 8, and Q is 1 to 4, wherein said oligonucleotide modulates mammalian telomere length.

12. The oligonucleotide of claim 11 which has at least one phosphorothioate linkage.

13. The oligonucleotide of claim 11 which has at least one ₂′

modification of a sugar of said oligonucleotide.

14. The oligonucleotide of claim 11 which is a chimeric oligonucleotide.