Drug – bio-affecting and body treating compositions – Designated organic active ingredient containing – Carbohydrate doai
Reexamination Certificate
2000-03-10
2001-04-03
Schwartzman, Robert A. (Department: 1635)
Drug, bio-affecting and body treating compositions
Designated organic active ingredient containing
Carbohydrate doai
C435S006120, C435S091100, C435S325000, C435S375000, C536S023100, C536S024310, C536S024330, C536S024500
Reexamination Certificate
active
06211164
ABSTRACT:
BACKGROUND OF THE INVENTION
1. Technical Field
The subject invention relates to the construction of antisense oligonucleotides of the human Chk1 gene and to uses thereof. The human Chk1 gene is a major G2/M checkpoint gene that is activated in response to DNA damage. In particular, the gene transduces the inhibitory signal from DNA damage sensors to the basic cell cycle machinery. Thus, antisense oligonucleotides to the human Chk1 gene may be used to inhibit gene expression thereby preventing G2 arrest induced by DNA damaging agents. Additionally, antisense oligonucleotides may act to sensitize tumor cells thereby making them more sensitive to therapy than normal cells.
2. Background Information
Many cancer therapeutic reagents cause cell death by inducing severe cellular DNA damage. Such DNA damage elicits two responses in normal, eukaryotic cells: 1) cell cycle arrest and 2) DNA repair to maintain genetic fidelity. In particular, checkpoint genes are activated in response to DNA damage. The gene products of these checkpoint genes form signal transduction pathways that transduce inhibitory signals from the DNA damage sensors to the basic cell cycle machinery and result in cell cycle arrest at both the G1 and G2 phases. Simultaneously, DNA damage also induces activation of transcription and production enzymes that facilitate DNA repair (Paulovich et al.,
Cell
88:315-321 (1997))).
p53 is the major G1 phase checkpoint gene. This protein is activated in the event of DNA damage (Shieh et al.,
Cell
91:325-34 (1997); Kastan et al.,
Cancer Research
51:6304-11 (1991)). (It transcriptionally activates cell cycle inhibitors such as p21, which, in turn, inhibit G1 cyclin-Cdks, thereby preventing cells from traversing the G1/S boundary (el-Deiry et al.,
Cancer Research
54:1169-74 (1994); Dulic et al.,
Cell
76:1013-1023 (1994)).
At the G2/M boundary, the onset of mitosis depends on the active cyclin B-Cdc2 kinase complex (King et al.,
Cell
79:563-71 (1994); Lew et al.,
Current Opinion in Cell Biology
8:795-804 (1996)). Weel kinase and Cdc25C phosphatase regulate Cdc2 activity. In particular, Weel phosphorylates Cdc2 at tyrosine 15 which inactivates Cdc2. Cdc25C removes this inhibitory phosphate and keeps Cdc2 active (Nurse et al.,
Cell
91: 865-7 (1997)).
During the G2 phase, DNA damages leads to Chk1 phosphorylation and activation in an ATM dependent manner (Walworth et al.,
Science
271:353-6 (1996)). Active Chk1 phosphorylates Cdc25C at serine 216, and 14-3-3 proteins export the phosphorylated Cdc25c out of the nucleus of the cell. Thus, Cdc2 is inhibited by the phosphorylation at tyrosine 15, and cells are halted at G2 for DNA repair (Furnari et al.,
Science
277:1495-7 (1997); Furnari et al.,
Molecular Biology of the Cell
10:833-45 (1999); Sanchez et al.,
Science
277:1497-501 (1997); Peng et al.,
Science
277:1501-5 (1997)).
It has been known for some time that the majority of tumors have defects in G1 checkpoint machinery, many of them due to p53 mutations (Kastan et al. (1991) Cancer Research 51:6304-11). In the event of DNA damage, these G1 checkpoint-defective tumor cells depend primarily on the G2 checkpoint for DNA repair. The inability to repair DNA at the G1 checkpoint is consistent with the observation that tumor cells are more sensitive to DNA damaging therapeutics than are normal cells. Given this observation, the toxicity to normal tissues is still a common side effect in cancer therapy. Significant effort has therefore been expended to specifically sensitize tumors to cancer drugs or radiotherapy.
One such approach is to abrogate G2 arrest in response to DNA damage (Powell et al.,
Cancer Research
55: 1643-8 (1995); Yao et al.,
Nature Medicine
2:1140-3 (1996)). Since G1 checkpoint-defective tumor cells can only repair DNA in G2 phase, inhibition of the Chk1 gene is expected to a brogate the G2 arrest in DNA damage response and increase the sensitivity to chemotherapy/radiotherapy more profoundly in tumor cells than in normal cells.
All U.S. patents and publications are herein incorporated in their entirety by reference.
SUMMARY OF THE INVENTION
The subject invention encompasses an isolated antisense nucleotide sequence of a mammalian Chk1 gene which inhibits expression of Chk1 protein. This sequence may be represented by SEQ ID NO:1 (oligo 7), SEQ ID NO:2 (oligo 8), SEQ ID NO:3 (oligo 9), SEQ ID NO:4 (oligo 14), or a fragment thereof which specifically hybridizes to the complement of one of these sequences. The sequence may also have at least 40% identity to SEQ ID NO:1, SEQ ID NO:2, SEQ ID:3 or SEQ:4, or it may be a fragment thereof which hybridizes to the complement of the sequence having 40% identity to the sequence having at least 40% identity to SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3 or SEQ ID NO:4.
Additionally, the present method includes a method of preventing expression of Chk1 protein by a cell comprising the step of introducing into the cell a vector comprising at least one of the above nucleotide sequences or fragments thereof.
Furthermore, the present invention also encompasses a method of preventing expression of Chk1 protein by a cell comprising the step of introducing into the cell a vector comprising an isolated nucleotide sequence having at least 40% identity to a nucleotide sequence selected from the group consisting of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, and a fragment thereof which specifically hybridizes to the complement of the isolated nucleotide sequence.
The present invention also includes a method of screening a compound for ability to inhibit expression of Chk1 protein by a cell comprising the steps of exposing the cell to the compound of interest and measuring expression of Chk1 protein by the cell, lack of expression of Chk1 protein indicating a compound having the ability to inhibit expression of Chk1 protein.
Additionally, the invention also encompasses a pharmaceutical composition comprising an isolated antisense nucleotide sequence of a mammalian Chk1 gene or homologue thereof which inhibits expression of a Chk1 protein and a pharmaceutically acceptable carrier.
The present invention also includes a pharmaceutical composition comprising: 1) an isolated nucleotide sequence having at least 40% identity to a nucleotide sequence selected from the group consisting of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3 and SEQ ID NO:4 or 2) a fragment thereof which specifically hybridizes to the complement of the isolated nucleotide sequence, and 3) a pharmaceutically acceptable carrier. More than one of the sequences and/or fragments thereof may be included in the composition.
Additionally, the present invention encompasses a method of sensitizing malignant cells to chemotherapy, in a patient in need of such treatment, comprising the step of administering to the patient an effective amount of the pharmaceutical composition or compositions described above.
REFERENCES:
patent: WO99/11795 (1999-03-01), None
Paulovich et al.,Cell88:315-321 (1997).
Shieh et al.,Cell91:325-34 (1997).
Kastan et al.,Cancer Research51:6304-11 (1991).
el-Deiry et al.,Cancer Research54:1169-74 (1994).
Dulic et al.,Cell76:1013-1023 (1994).
King et al.,Cell79:563-71 (1994).
Lew et al.,Current Opinion in Cell Biology8:795-804 (1996).
Nurse et al.,Cell91:865-7 (1997).
Walworth et al.,Science271:353-6 (1996).
Furnari et al.,Science277:1495-7 (1997).
Furnari et al.,Molecular Biology of the Cell10:833-45 (1999).
Sanchez et al.,Science277:1497-501 (1997).
Peng et al.,Science277:1501-5 (1997).
Powell et al.,Cancer Research55:1643-8 (1995).
Yao et al.,Nature Medicine2:1140-3 (1996).
Andrea D. Branch, A good antisense molecule is hard to find, TIBS, 47-48, Feb. 1998.*
Stanley Crooke, Antisense Research and Applications, Chapter 1, Basic Principles of Antisense Therapeutics, Springer-Verlag Press, Berlin, Heidelberg, New York p. 3, Feb. 1998.
Giranda Vincent L.
Luo Yan
Rockow-Magnone Shayna K.
Abbott Laboratories
Becker Cheryl L.
Epps Janet L.
Schwartzman Robert A.
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