Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving nucleic acid
Reexamination Certificate
2001-03-26
2002-05-14
Horlick, Kenneth R. (Department: 1656)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving nucleic acid
C435S007100, C435S091200
Reexamination Certificate
active
06387629
ABSTRACT:
FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT
Not applicable.
BACKGROUND OF THE INVENTION
This invention is directed to the field of medical diagnostics and treatment. More particularly, it is directed to methods of diagnosing and treating endometriosis based on the pathologic up-regulation of cathepsin S in endometriotic tissue.
Endometriosis is a painful disorder that is characterized by the ectopic implantation of functioning endometrial tissue into the abdominal wall and the outer surface of various organs including, most commonly, the lower bowel, ovaries and fallopian tubes. P. Vigano et al. (1991)
Fertility and Sterility
56:894. Currently, endometriosis-specific genes have not been identified and the events relating to the development of endometriosis are poorly understood. However, several reports suggest that retrograde menstruation linked with abnormal immune function may play a role in establishing ectopic endometriotic lesions. T. Ishimaru and H. Masuzaki (1991)
Am. J. Obstet. Gynecol.
165:210-214. The identification of genes that are differentially expressed in endometriotic lesions compared to healthy endometrial tissue would provide markers for diagnosing endometriosis and targets for therapeutic intervention in endometriosis.
SUMMARY OF THE INVENTION
Human endometrial tissue cultured in mice grows and mimics the progression to endometriosis. We have discovered that cathepsin S expression is up-regulated in such tissue. This invention provides methods and materials that take advantage of this fact. More particularly, this invention provides methods of diagnosing endometriosis by detecting up-regulation of cathepsin S in a sample from a patient suspected of having endometriosis. The methods involve detecting increased amounts of cathepsin S mRNA or cathepsin S protein in the sample compared to normal. This invention also provides methods of treating endometriosis by down-regulating the level of cathepsin S activity in ectopic or eutopic endometrial tissue. These methods include decreasing transcription, processing or translation of cathepsin S mRNA, as well as inhibiting biological activity of cathepsin S.
In one aspect this invention provides a method for use in the diagnosis of endometriosis in a subject. The method comprises the steps of: detecting a test amount of a cathepsin S gene product in a sample from the subject; and comparing the test amount with a normal amount of the cathepsin S gene product in a control sample. A test amount above the normal amount provides a positive indication in the diagnosis of endometriosis. In one aspect, the method comprises ectopic endometrial tissue, eutopic endometrial tissue, peritoneal fluid, blood, vaginal secretion or urine.
In one embodiment of the method, the cathepsin S gene product is cathepsin S mRNA or cDNA. The step of detecting can comprise the steps of contacting the cathepsin S mRNA or cDNA with a polynucleotide of at least 7 to about 50 nucleotides in length that specifically hybridizes to the cathepsin S mRNA or cDNA and detecting hybridization between the polynucleotide and the mRNA or cDNA. In one embodiment, the polynucleotide is a primer and the step of detecting hybridization comprises initiating reverse transcription of cathepsin S mRNA with the primer, and detecting a cathepsin S mRNA reverse transcript. Detection of the reverse transcript indicates that the polynucleotide has specifically hybridized to cathepsin S mRNA. In another embodiment the cathepsin S mRNA or cDNA is immobilized and the step of contacting comprises contacting the immobilized mRNA or cDNA with the polynucleotide. In another embodiment the polynucleotide is immobilized and the step of contacting comprises contacting the immobilized polynucleotide with the cathepsin S mRNA or cDNA. In another embodiment the biological sample is a fixed tissue sample and the step of contacting comprises contacting the polynucleotide with the mRNA or cDNA in situ on the fixed tissue sample.
In another embodiment the step of detecting comprises the steps of amplifying the cathepsin S mRNA or cDNA to produce an amplification product and detecting the amplification product. In one method, the step of detecting the amplification product comprises contacting the amplification product with a polynucleotide of at least 7 to about 50 nucleotides in length that specifically hybridizes to the amplification product, and detecting hybridization between the polynucleotide and the amplification product. In another method, the step of detecting the amplification product comprises determining the nucleotide sequence of the amplification product. In another method, the step of detecting the amplification product comprises determining the mass of the amplification product.
In another embodiment, the cathepsin S gene product is cathepsin S polypeptide. In one method, the step of detecting comprises detecting binding cathepsin S polypeptide by immunoassay. The immunoassay can be non-competitive or competitive.
A competitive immunoassay comprises detecting binding between the cathepsin S polypeptide and an antibody comprising a detectable moiety, e.g., selected from the group consisting of a fluorescent label, a radioactive label, an enzymatic label, a biotinyl group, or an epitope recognized by a secondary reporter. A non-competitive immunoassay comprises the steps of capturing the cathepsin S polypeptide from the sample on a solid phase with a first antibody specific for cathepsin S polypeptide; and detecting capture of the cathepsin S polypeptide by contacting the solid phase with a second antibody specific for cathepsin S polypeptide and detecting binding between the second antibody and cathepsin S polypeptide. Another non-competitive immunoassay comprises the steps of binding the cathepsin S polypeptide from the sample to a solid phase; and detecting the cathepsin S polypeptide by contacting the solid phase with an antibody specific for cathepsin S polypeptide and detecting binding between the antibody and cathepsin S polypeptide.
In another embodiment the method involves detecting the polypeptide by contacting the sample with an affinity agent that binds to cathepsin S polypeptide and detecting binding between the affinity agent and the cathepsin S polypeptide. In another method, the step of detecting comprises detecting an analyte in the sample having the mass of cathepsin S polypeptide.
In another aspect this invention provides method for use in the monitoring the progress of endometriosis in a subject comprising the steps of detecting a first test amount of a cathepsin S gene product in a sample from the subject at a first time; detecting a second test amount of the cathepsin S gene product in a sample from the subject at a second, later time; and comparing the first test amount with the second test amount. An increase in the amount between the first time and the second time indicates progression of endometriosis and a decrease in the amount between the first time and the second time indicates remission of endometriosis.
In another aspect this invention provides a kit comprising a compound that binds a cathepsin S gene product and instructions to (1) use the compound for detecting cathepsin S in a patient sample, and (2) to diagnose endometriosis based on an elevated amount of the cathepsin S gene product in the sample compared with a normal amount of cathepsin S.
In another aspect this invention provides method for use in the diagnosis of endometriosis in a subject comprising detecting a cathepsin S gene product in endometrial tissue from the subject in vivo, whereby detection of the gene product provides a positive indication in the diagnosis of endometriosis. In one embodiment the method comprises administering to the subject a compound that specifically binds to a cathepsin S gene product and detecting binding between the compound and the cathepsin S gene product. In one embodiment of the method the compound comprises a gamma-emitting or positron-emitting radioisotope and binding is detected by detecting the radioisotope by camera imaging or Geiger cou
French Cynthia K.
Schneider Patrick
Yamamoto Karen K.
Horlick Kenneth R.
Reprogen, Inc.
Townsend and Townsend / and Crew LLP
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