Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving nucleic acid
Reexamination Certificate
1999-04-19
2001-02-13
Horlick, Kenneth R. (Department: 1656)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving nucleic acid
C435S091100, C435S091200, C549S354000, C564S427000
Reexamination Certificate
active
06187543
ABSTRACT:
TECHNICAL FIELD
The present invention relates, in general, to tristetraprolin (TTP) and, in particular, to methods of modulating levels of tumor necrosis factor &agr; (TNF&agr;) using TTP or nucleic acid sequences encoding same. The invention further relates to methods of screening compounds for their ability to inhibit TNF&agr; biosynthesis, processing or secretion.
BACKGROUND
Tumor necrosis factor alpha (TNF&agr;) is a potent cytokine that is released from many cell types, particularly, macrophages and monocytes. TNF&agr; also exists in a cell-membrane bound, higher molecular weight form on cells, and this form also appears to mediate a variety of biological effects. TNF&agr; is thought to have few roles in normal development and physiology; however, it exerts harmful and destructive effects on many tissues in many disease states (Tracey et al, Ann. Rev. Med. 45:491 (1994)). Disease states in which TNF&agr; has been shown to exert a major pathogenetic role include septic shock syndrome, cancer cachexia, rheumatoid arthritis, etc. Many investigators and pharmaceutical companies are actively investigating agents and potential drugs that can block TNF&agr; effects, either by blocking its synthesis or interfering with its binding to its surface receptors.
One example of this approach is the use of monoclonal antibodies to TNF&agr;. These have been used in animal models of human disease, and in human conditions such as rheumatoid arthritis (Arend et al, Arthritis and Rheumatism 38:151 (1995)). There is no question that these antibodies temporarily relieve some of the signs and symptoms of this disease in man. However, their potential widespread use is compromised by many factors, especially the fact that they seem to be only temporarily (ie, a few months) effective. Other drawbacks include expense, the need for parenteral administration, the likelihood that anti-idotype antibodies will develop, etc.
The present invention relates to a novel approach to the treatment of diseases the effects of which are mediated, at least in part, through TNF&agr;. This approach involves the protein tristetraprolin (TTP) and nucleic acid sequences encoding same.
TTP (Lai et al, J. Biol. Chem. 265:16556 (1990)), also known as Nup475 (DuBois et al, J. Biol. Chem. 265:19185 (1990)) and TIS11 (Varnum et al, Oncogene 4:119 (1989); Varnum et al, Mol. Cell. Biol. 11:1754 (1991)), is a widely distributed 33.6 kDa phosphoprotein encoded by the immediate-early response gene, Zfp-36 (Taylor et al, Nucl. Acids Res. 19:3454 (1991)). This gene has been mapped to chromosome 7 in the mouse, and the equivalent human gene, ZFP36, has been mapped to chromosome 19q 13.1 (Taylor et al, Nucl. Acids Res. 19:3454 (1991)). TTP is the prototype of a group of proteins containing two or more highly conserved putative zinc fingers of the CCCH class (Varnum et al, Mol. Cell. Biol. 11:1754 (1991); Taylor et al, Nucleic Acids Res. 19:3454 (1991); Gomperts et al, Oncogene 5:1081 (1990); Ma et al, Oncogene 9:3329 (1994)). In addition, the protein has been shown to bind Zn
++
and has been localized to the cell nucleus in mouse fibroblasts (DuBois et al, J. Biol. Chem. 265:19185 (1990)), suggesting that it may be a transcription factor. Serum or other mitogen stimulation of quiescent fibroblasts causes rapid serine phosphorylation and nuclear to cytosolic translocation of TTP (Taylor et al, J. Biol. Chem. 270:13341 (1995); Taylor et al, Mol. Endocrinol. 10:140 (1996)), both of which are likely to modulate its function in cells.
In the adult mouse, TTP mRNA is highly expressed in lung, intestine, lymph node, spleen, and thymus, with lower expression in adipose tissue, kidney, and liver, and negligible expression in skeletal muscle and brain (Lai et al, J. Biol. Chem. 265:16556 (1990); DuBois et al, J. Biol. Chem. 265:19185 (1990)). In the thymus, TTP mRNA is highly expressed in both cortical and medullary thymocytes, while in the spleen, it is highly expressed in B and T lymphocytes within the white pulp, and is expressed at somewhat lower levels in the myeloid cells of the red pulp and endothelial cells of the high endothelial venules. In addition, TTP is constitutively expressed in several types of blood cells, particularly neutrophils, macrophages and B and T lymphocytes. The function of TTP in normal vertebrate physiology, however, was heretofore unknown.
OBJECTS AND SUMMARY OF THE INVENTION
It is a general object of the invention to provide a method of modulating cellular levels of TNF&agr;.
It is a specific object of the invention to provide a method of treating diseases and disorders associated with TNF&agr; excess.
It is a further object of the invention to provide a method of identifying an individual at increased risk to the effects of TNF&agr; excess.
It is another object of the invention to provide a method of selecting compounds for there ability to inhibit TNF&agr; production, processing or secretion.
It is a further object of the invention to provide a TTP-deficient non-human mammal.
These objects are met by the present invention.
In one embodiment, the present invention relates to a method of inhibiting TNF&agr; production, processing or secretion in a mammal. The method comprises increasing the level of TTP, or a TNF&agr; production, processing or secretion-inhibitory polypeptide fragment thereof, in the mammal so that the inhibition is effected.
In a further embodiment, the present invention relates to a method of treating an effect of excess TNF&agr; in a mammal. The method comprises administering to the mammal TTP, or polypeptide fragment thereof that inhibits TNF&agr; production, processing or secretion, or agent that enhances a TNF&agr; production, processing or secretion-inhibitory effect of TTP, in an amount sufficient to effect the treatment.
In yet another embodiment, the present invention relates to a method of identifying a subject susceptible to a TNF&agr; associated disease or disorder. The method comprises isolating a DNA-containing biological sample from the subject, locating the TTP gene present in the DNA and comparing the nucleic sequence of the TTP gene with a wild-type TTP encoding sequence and thereby determining whether the TTP gene includes a mutation that renders the subject susceptible to the disease or disorder.
In a further embodiment, the present invention relates to a method of identifying a subject susceptible to a TNF&agr; associated disease or disorder. The method comprises
i) isolating a biological sample from the subject,
ii) contacting the sample with a TTP binding partner under conditions such that complexation between TTP and the binding partner can occur, and
iii) detecting the presence or absence of the complexation, or comparing the extent of the complexation with a control sample comprising wild-type TTP.
In another embodiment, the present invention relates to a method of screening a compound for its ability to enhance the ability of TTP to inhibit TNF&agr; production. The method comprises
i) contacting the compound with a sample comprising an expression construct comprising a TNF&agr; encoding sequence, in the presence of TTP or TNF&agr; production-inhibitory polypeptide fragment thereof, under conditions such that the TNF&agr; encoding sequence can be expressed, and
ii) determining the level of expression of the TNF&agr; encoding sequence and comparing that level to a level of expression obtained in the absence of the compound.
In yet a further embodiment, the present invention relates to a method of screening a compound for its ability to enhance a TNF&agr; transcription-repressor effect of TTP. The method comprises
i) contacting the compound with a sample comprising an expression construct comprising a TNF&agr; promoter sequence operably linked to an encoding sequence, in the presence of TTP or TNF&agr; production-inhibitory polypeptide fragment thereof, under conditions such that the encoding sequence can be expressed, and
ii) comparing the level of expression of the encoding sequence obtained to a level of expression obtained in the absence of the compound.
In
Duke University
Horlick Kenneth R.
Nixon & Vanderhye P.C.
Taylor Janell E.
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