Chemistry: molecular biology and microbiology – Animal cell – per se ; composition thereof; process of...
Reexamination Certificate
1995-06-07
2002-07-09
Kunz, Gary L. (Department: 1647)
Chemistry: molecular biology and microbiology
Animal cell, per se ; composition thereof; process of...
C536S023500, C536S023400, C435S252300, C435S320100
Reexamination Certificate
active
06416998
ABSTRACT:
BACKGROUND OF THE INVENTION
This invention relates to gene therapy whereby modified steroid receptors regulate the expression of genes within tissue.
Intracellular receptors are a superfamily of related proteins that mediate the nuclear effects of steroid hormones, thyroid hormone and vitamins A and D (Evans,
Science
240:889-895 (1988)). The cellular presence of a specific intracellular receptor defines that cell as a target for the cognate hormone. The mechanisms of action of the intracellular receptors are related in that they remain latent in the cytoplasm or nuclei of target cells until exposed to a specific ligand (Beato,
Cell
56:335-344 (1989); O'Malley, et al.,
Biol. Reprod
. 46:163-167 (1992)). Interaction with hormone then induces a cascade of molecular events that ultimately lead to the specific association of the activated receptor with other proteins or regulatory elements of target genes. The resulting positive or negative effects on regulation of gene transcription are determined by the cell-type and promoter-context of the target gene.
In the case of steroid hormones and steroid receptors, such complexes are responsible for the regulation of complex cellular events, including activation or repression of gene transcription. For example, the ovarian hormones, estrogen and progesterone, are responsible, in part, for the regulation of the complex cellular events associated with differentiation, growth and functioning of female reproductive tissues. Likewise, testosterone is responsible for the regulation of complex cellular events associated with differentiation growth and function of male reproductive tissues.
In addition, these hormones play important roles in development and progression of malignancies of the reproductive endocrine system. The reproductive steroids estrogen, testosterone, and progesterone are implicated in a variety of hormone-dependent cancers of the breast (Sunderland, et al.,
J. Clin. Oncol
. 9:1283-1297 (1991)), ovary (Rao, et al.,
Endocr. Rev
. 12:14-26 (1991)), endometrium (Dreicer, et al.,
Cancer Investigation
10:27-41, (1992)), and possibly prostate (Daneshgari, et al.,
Cancer
71:1089-1097 (1993)). In addition, the onset of post-menopausal osteoporosis is related to a decrease in production of estrogen (Barzel,
Am. J. Med
. 85:847-850 (1988)).
In addition, corticosteroids are potent and well-documented mediators of inflammation and immunity. They exert profound effects on the production and release of numerous humoral factors and the distribution and proliferation of various cellular components associated with the immune and inflammatory responses. For example, steroids are able to inhibit the production and release of cytokines (IL-1, IL-2, IL-3, IL-6, IL-8, TNF-&agr;, IFN-&ggr;), chemical mediators (eicosinoids, histamine), and enzymes (MMPs) into tissues, and directly prohibit the activation of macrophages and endothelial cells. Due to the global down-regulation of these physiological events, corticosteroids have a net effect of suppressing the inflammatory response and have therefore been used extensively to treat a variety of immunological and inflammatory disorders (rheumatoid arthritis, psoriasis, asthma, allergic rhinitis, etc.).
Besides the therapeutic benefits, however, there are some severe toxic side effects associated with steroid therapy. These include peptic ulcers, muscle atrophy, hypertension, osteoporosis, headaches, etc. Such side effects have hindered the utilization of steroids as therapeutic agents.
In general, the biological activity of steroid hormones is mediated directly by a hormone and tissue-specific intracellular receptor. Ligands are distributed through the body by the hemo-lymphatic system. The hormone freely diffuses across all membranes but manifests its biological activity only in those cells containing the tissue-specific intracellular receptor.
In the absence of ligand, the inactive steroid hormone receptors such as the glucocorticoid (“GR”), mineral corticoid (“MR”), androgen (“AR”) progesterone (“PR”) and estrogen (“ER”) receptors are sequestered in a large complex consisting of the receptor, heat-shock proteins (“hsp”) 90, hsp70 and hsp56 and other proteins as well. Smith, et al.,
Mol. Endo
. 7:4-11 (1993). The cellular localization of the physiologically inactive form of the oligomeric complex has been shown to be either cytoplasmic or nuclear. Picard, et al.,
Cell Regul
. 1:291-299 (1992); Simmons, et al.,
J. Biol. Chem
. 265:20123-20130 (1990).
Upon binding its agonist or antagonist ligand, the receptor changes conformation and dissociates from the inhibitory heteroligomeric complex. Allan, et al.,
J. Biol. Chem
. 267:19513-19520 (1992); Allan, et al.,
P.N.A.S
. 89:11750-11754 (1992). In the case of GR and other related systems such as AR, MR, and PR, hormone binding elicits a dissociation of heat shock and other proteins and the release of a monomeric receptor from the complex. O'Malley, et al.,
Biol. Reprod
. 46:163-167 (1992). Studies from genetic analysis and in vitro protease digestion experiments show that conformational changes in receptor structure induced by agonists are similar but distinct from those induced by antagonists. Allan, et al.,
J. Biol. Chem
. 267:19513-19520 (1992); Allan, et al.,
P.N.A.S
. 89:11750-11754 (1992); Vegeto, et al.,
Cell
69:703-713 (1992). However, both conformations are incompatible with hsp-binding.
Following the conformation changes in receptor structure, the receptors are capable of interacting with DNA. Studies suggest that the DNA binding form of the receptor is a dimer. In the case of GR homodimers, Tsai, et al.,
Cell
55:361-369 (1988), this allows the receptor to bind to specific DNA sites in the regulatory region of target gene promoters. Beato,
Cell
56:335-344 (1989). These short nucleotide sketches are arranged as palindromic, inverted or repeated repeats. Id. Specificity is determined by the sequence and the spacing of the repeated sequences. Umesono, et al.,
Cell
57:1139-1146. Following binding of the receptor to DNA, the hormone is responsible for mediating a second function that allows the receptor to interact specifically with the transcription apparatus. Such interaction could either provide positive or negative regulation of gene expression, i.e., steroid receptors are ligand-binding transcription factors, capable of not only activating but also repressing the expression of specific genes. Studies have shown, however, that repression does not require DNA binding.
For instance, when bound to their intracellular receptors, corticosteroids can affect the transcription of a variety of genes whose products play key roles in the establishment and progression of an inflamed situation. Such genes include those encoding for cytokines, chemical mediators and enzymes. Transcription of these genes can be repressed or activated depending on the transcription factors and/or regulatory sequences controlling the expression of the gene. Presently there are numerous reports documenting the effect of glucocorticoid on the expression of various genes at the transcriptional level.
In particular, the glucocorticoid receptor is a member of a family of ligand-dependent transcription factors capable of both positive and negative regulation of gene expression (Beato,
FASEB J
. 5:2044-2051 (1991); Pfahl,
Endocr. Rev
. 14:651-658, (1993); Schule, et al.,
Trends Genet
. 7:377-381 (1991)). In its inactivated form, the GR is part of a large heteromeric complex which includes hsp90 as well as other proteins (Denis, et al.,
J. Biol. Chem
. 262:11803-11806 (1987); Howard, et al.,
J. Biol. Chem
. 263:3474-3481 (1988); Mendel, et al.,
J. Biol. Chem
. 261:3758-3763 (1986); Rexin, et al.,
J. Biol. Chem
. 267:9619-9621 (1992); Sanchez, et al.,
J. Biol. Chem
. 260:12398-12401 (1985)), and hsp56 (Lebea, et al.,
J. Biol. Chem
. 267:4281-4284 (1992); Pratt,
J. Steroid Biochem. Mol. Biol
. 46:269-279 (1993); Rexin,
J. Biol. Chem
. 267:9619-9621 (1992); Sanchez,
J. Biol. Chem
. 265:22067-22070 (1990); Yem,
J. Biol. Chem
. 267:2868-2871,
Kittle, Jr. Joseph D.
Ledebur, Jr. Harry C.
O'Malley Bert W.
Tsai Ming-Jer
Baylor College of Medicine
Hayes Robert C.
Kunz Gary L.
Lyon & Lyon LLP
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