Claims
- 1. A method of inhibiting Mycobacterium tuberculosis glutamine synthetase protein expression comprising contacting a Mycobacterium tuberculosis bacterium with an effective amount of an antisense compound comprising an antisense polynucleotide that hybridizes to a Mycobacterium tuberculosis glutamine synthetase polynucleotide, wherein the antisense polynucleotide hybridizes to a region of the Mycobacterium tuberculosis glutamine synthetase polynucleotide encoding the glutamine synthetase protein, thereby inhibiting Mycobacterium tuberculosis glutamine synthetase protein expression.
- 2. The method of claim 1, wherein the polynucleotide has modification to its internucleoside phosphates linkages selected from the group consisting of phosphorothioates, methylphosphonates, phosphoroboronates, phosphoromorpholidates, butyl amidates, and peptide nucleic add linkages.
- 3. The method of claim 2, wherein the polynucleotide is selected from the group consisting of 5′-GAC GTC GTC GGG CGT CTT-3′ (SEQ ID NO: 18), 5′-CAT GCC GGA CCC GTT GTC GCC-3′ (SEQ ID NO: 19) and 5′-CCA CAG CGA CTG ATG ACA GTG CAT-3′ (SEQ ID NO: 20).
- 4. The method of claim 1, further comprising contacting the Mycobacterium tuberculosis with an effective amount of an antibiotic capable of inhibiting the proliferation of Mycobacterium tuberculosis.
- 5. The method of claim 4, wherein the antibiotic is selected from the group consisting of rifampin, isoniazid, amikacin, ethambutol and polymyxin B nonapeptide.
- 6. The method of claim 1, further comprising contacting the Mycobacterium tuberculosis with an effective amount of a second antisense polynucleotide that hybridizes to a Mycobacterium tuberculosis glutamine synthetase polynucleotide, wherein the second antisense polynucleotide hybridizes to a region of the Mycobacterium tuberculosis glutamine synthetase polynucleotide encoding the glutamine synthetase protein that is distinct from the region targeted by the antisense polynucleotide of claim 1.
- 7. The method of claim 1, further comprising contacting the Mycobacterium tuberculosis with an effective amount of a second antisense polynucleotide that hybridizes to a Mycobacterium tuberculosis polynucleotide selected from the group consisting of polynucleotides that encode AroA, Ask, GroES or the 30, 32A, 32B extracellular proteins.
- 8. The method of claim 1, wherein the antisense polynucleotide inhibits the growth of Mycobacterium tuberculosis.
- 9. An antisense compound of about 15 to about 50 nucleobases in length targeted to a portion of a nucleic acid molecule encoding a Mycobacterium tuberculosis glutamine synthetase protein, wherein the antisense compound specifically hybridizes with the nucleic acid molecule encoding the Mycobacterium tuberculosis glutamine synthetase protein, thereby inhibiting the expression of the Mycobacterium tuberculosis glutamine synthetase protein.
- 10. The antisense compound of claim 9 which is an antisense oligonucleotide.
- 11. The antisense compound of claim 9, wherein the antisense polynucleotide comprises 5′-GAC GTC GTC GGG CGT CTT-3′ (SEQ ID NO: 18), 5′-CAT GCC GGA CCC GTT GTC GCC-3′ (SEQ ID NO: 19) or 5′-CCA CAG CGA CTG ATG ACA GTG CAT-3′ (SEQ ID NO: 20).
- 12. The antisense compound of claim 9 which comprises at least one modified internucleoside linkage.
- 13. The antisense compound of claim 12, wherein the modified internucleoside linkage is a phosphorothioate linkage.
- 14. The antisense compound of claim 9, wherein the antisense polynucleotide has a complete homology with the nucleic acid molecule encoding the Mycobacterium tuberculosis glutamine synthetase protein.
- 15. A process for producing an antisense compound that inhibits the expression of Mycobacterium tuberculosis glutamine synthetase comprising the steps of:
(a) synthesizing a antisense polynucleotide of about 15 to about 50 nucleobases in length, wherein the antisense polynucleotide is capable of hybridizing to a portion of a Mycobacterium tuberculosis polynucleotide encoding a Mycobacterium tuberculosis glutamine synthetase protein; (b) contacting a culture of Mycobacterium tuberculosis with an effective amount of the antisense polynucleotide of step (a) such that the antisense polynucleotide hybridizes with a Mycobacterium tuberculosis glutamine synthetase polynucleotide; (c) comparing the levels of Mycobacterium tuberculosis glutamine synthetase protein expression in the culture contacted with the effective amount of the antisense polynucleotide to the levels of Mycobacterium tuberculosis glutamine synthetase protein expression in a control culture of Mycobacterium tuberculosis, wherein the control culture is not contacted with the antisense polynucleotide; and (d) determining whether the antisense polynucleotide inhibits the expression of glutamine synthetase by observing the level of glutamine synthetase expression in the Mycobacterium tuberculosis culture contacted with the antisense polynucleotide relative to the level of glutamine synthetase expression in the Mycobacterium tuberculosis control culture.
- 16. An antisense polynucleotide produced according to the process of claim 15.
- 17. A method of inhibiting protein expression of a Mycobacterium tuberculosis gene selected from the group consisting of AroA, Ask, GroES or the 30, 32A, 32B extracellular proteins comprising contacting Mycobacterium tuberculosis with an effective amount of an antisense polynucleotide that hybridizes to a Mycobacterium tuberculosis AroA, Ask, GroES, 30, 32A or 32B extracellular protein polynucleotide, wherein the antisense polynucleotide hybridizes to a region of the Mycobacterium tuberculosis polynucleotide encoding the protein.
- 18. An antisense compound of about 15 to about 50 nucleobases in length targeted to a portion of a nucleic acid molecule encoding a Mycobacterium tuberculosis protein selected from the group consisting of AroA, Ask, GroES, 30, 32A and 32B extracellular proteins, wherein the antisense compound specifically hybridizes with nucleic acid molecule encoding a Mycobacterium tuberculosis AroA, Ask, GroES, 30, 32A or 32B extracellular protein, thereby inhibiting the expression of the Mycobacterium tuberculosis AroA, Ask, GroES, 30, 32A or 32B extracellular protein.
- 19. A process for producing an antisense compound that inhibits the expression of a Mycobacterium tuberculosis gene selected from the group consisting of AroA, Ask, GroES or 30, 32A, 32B extracellular proteins comprising the steps of.
(a) synthesizing a antisense polynucleotide of about 15 to about 50 nucleobases in length, wherein the antisense polynucleotide is capable of hybridizing to a portion of polynucleotide encoding a Mycobacterium tuberculosis AroA, Ask, GroES or 30, 32A, 32B extracellular proteins; (b) contacting a culture of Mycobacterium tuberculosis with an effective amount of the antisense polynucleotide such that the antisense polynucleotides hybridizes to a Mycobacterium tuberculosis aroA, ask, groES or 30, 32A, 32B extracellular protein polynucleotide; (c) comparing the levels of Mycobacterium tuberculosis AroA, Ask, GroES or 30, 32A, 32B extracellular protein expression in the culture contacted with the effective amount of the antisense polynucleotide to the levels of Mycobacterium tuberculosis AroA, Ask, GroES or 30, 32A, 32B extracellular protein expression in a control culture of Mycobacterium tuberculosis, wherein the control culture is not contacted with the antisense polynucleotide; and (d) determining whether the antisense polynucleotide inhibits the expression of AroA, Ask, GroES or 30, 32A, 32B extracellular proteins by observing the level of glutamine synthetase expression in the Mycobacterium tuberculosis culture contacted with the effective amount of the antisense polynucleotide relative to the level of AroA, Ask, GroES or 30, 32A, 32B extracellular protein expression in the Mycobacterium tuberculosis control culture.
- 20. An antisense polynucleotide produced according to the process of claim 19.
- 21. A method of inhibiting Mycobacterium tuberculosis protein expression comprising contacting a Mycobacterium tuberculosis bacterium with an effective amount of an antisense compound comprising an antisense polynucleotide that hybridizes to a Mycobacterium tuberculosis polynucleotide, wherein the antisense polynucleotide hybridizes to a region of the Mycobacterium tuberculosis polynucleotide encoding a Mycobacterium tuberculosis protein, thereby inhibiting the expression of the protein.
- 22. The method of claim 21, wherein the protein is an extracellular protein.
Parent Case Info
[0001] This application claims the benefit of U.S. provisional patent application No. 60/171,929, filed Dec. 22, 1999, the entire contents of which are incorporated herein by reference.
Government Interests
[0002] This invention was made with Government support under Grant Nos. AI 31338 and AI 42925, awarded by the National Institutes of Health. The Government has certain tights in this invention.
PCT Information
Filing Document |
Filing Date |
Country |
Kind |
PCT/US00/34688 |
12/20/2000 |
WO |
|