Chemistry: molecular biology and microbiology – Micro-organism – tissue cell culture or enzyme using process... – Preparing compound containing saccharide radical
Reexamination Certificate
1999-01-12
2003-03-25
Guzo, David (Department: 1636)
Chemistry: molecular biology and microbiology
Micro-organism, tissue cell culture or enzyme using process...
Preparing compound containing saccharide radical
C435S006120, C435S029000, C435S320100, C435S455000, C435S091400, C536S023100, C536S023200, C536S024100
Reexamination Certificate
active
06537784
ABSTRACT:
TECHNICAL FIELD OF THE INVENTION
The present invention relates to the fields of molecular biology and immunology. More specifically, this invention relates to the induction of apoptosis in inflammatory cells by introducing into those cells a gene which induces apoptosis (programmed cell death or non-necrotic cell death) in these cells.
BACKGROUND OF THE INVENTION
In many inflammatory conditions, cytokines such as IL-1&bgr;, IL-10, GM-CSF and TNF&agr; are excessively produced as a result of mass aggregation and accumulation of inflammatory cells (Brennan F. M. et al.,
British Medical Bulletin
1995, 51/2, 368-384). Upregulation and/or dysregulation of cytokines in inflamed tissue may be directly or indirectly responsible for exacerbation of chronic inflammatory diseases. For example, the most marked pathology in rheumatoid arthritis (RA) is displayed at the local site of inflammation (i.e., the synovial joints). Therefore, it is likely that the cytokines produced in the synovial joints of RA patients play an important role in the disease process. Of those cytokines, IL-1&bgr; and TNF&agr; are believed to be responsible for the devastating cartilage destruction and bone erosion which characterizes RA (Dayer J. M. et al.,
J. Exp. Med
., 1985, 162, 1208-1215; Gowen M. et al.,
Nature
, 1983, 306, 378-380). The presence of excessive amounts of IL-1&bgr; and TNF&agr; in the synovial joints has been shown to accelerate development of collagen-induced arthritis in rodents (Brennan F. M., et al.,
Clin. Expt. Immunol
., 1994, 97/1, 1-3).
Apoptosis is a fundamental physiological process for embryonic development and maintenance of tissue homeostasis. (Raff, M. C. Nature, 1992, 356, 397; Vaux, D. L. et al. Cell, 1994, 76, 777). Inconsistency in this critical natural process is featured in a variety of neoplastic, neurodegenerative and autoimmune diseases (Thompson, C. B., Science, 1995, 267, 1456). Biochemical attributes, involving signal transduction cascade, are relatively complex and are not completely understood. A variety of stimuli including activation of specific receptors, such as TNFR1 or Fas trigger evolutionary conserved execution machinery involving several signaling components, that are orchestrated to cause cellular demise (Ashkenazi, A. and Dixit, V. M., Science, 1998, 181, 1305).
Granzyme B is a serine protease, primarily found in cytoplasmic granules of cytotoxic T lymphocytes and natural killer cells. Granzyme B plays an important role in inducing apoptotic changes in target cells by cytotoxic cell mediated killing (Huesel J. W. et al, Cell, 76, 977-987, 1994; Shi, L. et al. J. Exp. Med. 176,1521-1529, 1992), partly by catalyzing cleavage and activation of several caspases (Salvesen, G. S. and Dixit, V. M., Cell, 91, 443-446, 1997) as well as by caspase independent pathways (Andrade, F. et al. Immunity 8,451-460,1998). Structurally, Granzyme B is produced as polypeptide containing a leader peptide separated by an inactivating di-peptide (Gly-Glu) from the active Granzyme B polypeptide. Like the caspases, Granzyme B recognizes substrates specifically at aspartic acid for cleavage.
TNF&agr; is a cytokine, mainly synthesized by monocytes, macrophages and lymphocytes in response to activation. The classic elements governing its expression are located in the proximal or the distal promoter region (reviewed in Pauli, U. Critical Rev. in Eukaryotic Gene Expression, 1994, 4, 323-344). Summarized below are regions that have been described to play a significant role in the TNF&agr; promoter activity:
a) TNF&agr;-responsive elements were shown to be located between base pairs −100 to −125. The region −108 to −101bp contains a palindrome, TGAGCTCA , which is similar to AP-1 sequence that contains PMA-responsive elements. Multiple copies of −125 to −85bp confer a 7 to 11 fold induction of the expression of the reporter gene (Leitman, D. et al, J. Biol. Chem. 266, 9343, 1991).
b) PMA-responsive elements were shown to be present in between −101 to −286 base pairs (Hensel, G. et al, Lymphokine Res. 8, 347, 1989).
c) Anti-CD3 antibody-induced (as well as Ca-ionophore- induced) responsive elements were shown to lie between −118 to −80 base pairs. The KappaB3 (GGGTTTCTCC) SEQ ID NO: 15 sequence in this region is of high importance for CsA-sensitive activation of the TNF&agr; promoter by Ca-ionophore. These elements are suggested to be optimally functional in the context of their own promoter (Goldfield, et al. J. Exp. Med. 178, 1356, 1993).
d) In U937 cells, the PMA responsive element is located between −95 to −36bp and the cAMP-responsive element (CRE) is mapped to position −107 to-99bp. This region does not respond to PMA (Economou, J. S. et al , J. Exp. Med. 170, 321, 1989).
e) All three kappaB sites [viz. kappaB1 (−587 to −577), kappaB2 (−210 to −202) and kappaB3 (−98 to −87) ]bound virus-inducible protein, although deletion of these sites did not affect virus inducibility (Goldfield, A. et al, PNAS, 87, 9769, 1990). Further more, deletion mutants of kappaB sites show that they are not primary targets for PMA stimulation of human TNF&agr; gene (Goldfield, A. et al J. Exp. Med. 174, 73, 1991).
f) In the murine system, the TNF&agr; promoter constructs −1059, −695 and −655bp are strongly LPS inducible. This LPS-inducibility was greatly reduced in a −451bp construct and further between −301 and −241bp. The −1059bp fragment of TNF&agr; promoter was silent in macrophages and was strongly expressed after LPS stimulation. The largest drop of activation was at −695 to −655bp, which contains a kappaB element in the murine TNF&agr; promoter (Shakhov, A. N. et al, J. Exp. Med., 1990, 171,35; Drouet, C. et al, J. Immunol., 1991, 147, 1694).
Elements in the 3′ untranslated region (3′UTR) of the TNF&agr; gene are known to be important for post-transcriptional regulation. Analysis of the influence of 3′ UTR has been made in the murine system, wherein conjunction with the homologous promoter, LPS inducibility was very strong. Using murine TNF&agr; promoter system, it was shown that 3′UTR effectively inhibits CAT activity in three non-macrophage cell lines viz. HeLa, NIH3T3 and L929. The sequence TTATTTAT was repeated several times in the 3′UTR and was proposed to be involved in regulation (Han, J., et al.,
J. Immunology
, 1991, 146, 1843-1848; Crawford, F. K., et al.,
J. Biol. Chem
., 1996, 271, 22383-22390).
A variety of cells such as activated macrophages, activated T cells, macrophage-like synoviocytes as well as fibroblasts-like synoviocytes, and transformed macrophage-like synoviocytes (also referred to as pannocyte) are present in the inflamed joints. An invasive structure, called the pannus, derives from the hyperplastic nature of synoviocytes and pannocytes. The pannus may result from an excessive proliferation of cells and/or diminished apoptosis in these cells. Proliferative index in these cells was shown to be relatively low. Therefore, hyperplasia in synoviocytes could be due to abnormalities in apoptosis of the synovial lining. The frequency of cells with end stage apoptosis is low in the synovium. Abnormalities of p53 mutations, which could result in resistance to apoptosis are reported in the RA synovial fibroblasts. Additionally, excessive amounts of pro-inflammatory cytokines such as TNF&agr; and IL-1&bgr; are produced in the synovial tissue by a variety of cell types at the cartilage-pannus junction, including cells of the macrophage lineage, macrophage-like synoviocytes, activated T-cells and possibly fibroblast-like synoviocytes (Chu C. Q. et al.,
Arthritis & Rheumatism
, 1991, 34, 1125-1132; Deleuran B. W., et al.,
Arthritis & Rheumatism
, 1992, 35, 1170-1178). This perpetuates the infiltration of inflammatory cells and production of more pro-inflammatory cytokines and factors, which are responsible for synovial cell proliferation. In addition to the above described inf
Barton Randall W.
Marlin Steven D.
Tatake Revati J.
Boehringer Ingelheim Pharmaceuticals Inc.
Devlin Mary-Ellen M.
Guzo David
Leffers, Jr. Gerald G.
Pocchiari Susan K.
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