Drug – bio-affecting and body treating compositions – Designated organic active ingredient containing – Cyclopentanohydrophenanthrene ring system doai
Reexamination Certificate
1998-05-14
2001-12-25
Clark, Deborah J. R. (Department: 1633)
Drug, bio-affecting and body treating compositions
Designated organic active ingredient containing
Cyclopentanohydrophenanthrene ring system doai
C435S320100, C435S325000, C435S455000, C536S023100, C536S023400
Reexamination Certificate
active
06333318
ABSTRACT:
FIELD OF THE INVENTION
The present invention relates to methods in the field of recombinant DNA technology, and products related thereto. More particularly, the invention relates to methods and products for modulating the expression of exogenous genes in mammalian systems.
BACKGROUND OF THE INVENTION
The steroid/thyroid hormone receptors comprise a superfamily of ligand-dependent transcription factors that play a crucial role in mediating changes in cell fate and function (Evans, R. M.,
Science
240:889-895 (1988)). The receptors transduce extracellular hormonal signals to target genes that contain specific enhancer sequences referred to as hormone response elements (HREs) Evans, (1988); Green and Chambon,
Trends Genet.
4:309-314 (1988); Yamamoto, K. R.,
Annu. Rev. Genet.
19:209-252 (1985)). Each receptor recognizes its own HRE, assuring that a distinct response is triggered by each hormonal signal. Together the collection of related transcription factors and their cognate response elements provides a unique opportunity to control gene expression.
The DNA binding domain of each member of the steroid/thyroid hormone superfamily of receptors has 66-68 amino acids. Twenty of these, including nine cysteines, are conserved throughout the family. The modular structure of members of this receptor superfamily allows the exchange of homologous domains between receptors to create functional chimeras. This strategy was used to demonstrate that the DNA binding domain is solely responsible for the specific recognition of the HRE in vivo (Green and Chambon,
Nature
325:75-78 (1987); Giguére et al.,
Nature
330:624-629 (1987); Petkovich et al.,
Nature
330:444-450 (1987); Kumar et al.,
Cell
51:941-951 (1987); Umesono et al.,
Nature
336:262-265 (1988); Thompson and Evans,
Proc. Natl. Acad. Sci. U.S.A.
86:3494-3498 (1989) and in vitro (Kumar and Chambon,
Cell
55:145-156 (1988)). By analogy with the proposed structure for Xenopus transcription factor IIIA (Miller et al.,
EMBO J.
4:1609-1614 (1985)), the invariant cysteines are thought to form two “zinc finger” that mediate the DNA binding function (Hollenberg and Evans,
Cell
55:899-906 (1988)). Involvement of these cysteines in Zn(II) coordination is supported by extended X-ray absorption fine structure (Freedman et al.,
Nature
334:543-546 (1988)), and by the effect of point mutagenesis experiments on DNA binding (Hollenberg and Evans, (1988)); Severne et al.,
EMBO J.
7:2503-2508 (1988)).
The HREs are in fact structurally related but functionally distinct. The glucocorticoid receptor response element (GRE), estrogen receptor response element (ERE), and thyroid hormone receptor response element (TRE) have been characterized in detail. These particular response elements have been found to have a palindromic pair of hexameric “half-sites” (Evans, (1988); Green and Chambon, (1988)). With optimized pseudo- or consensus response elements, only two nucleotides per half-site differ between GRE and ERE (Klock et al.,
Nature
329:734-736 (1987)). On the other hand, EREs and TREs have identical half-sites but the number of nucleotide spacers between the two half sites is different (Glass et al.,
Cell
54:313-323 (1988)).
In contrast to response elements having the palindromic sequence motif, the following hormone receptors typically recognize response elements having two half-sites in a direct-repeat (DR) sequence motif: RXR, RAR, COUP-TF, PPAR, and the like (see, e.g., Mangelsdorf et al.,
The Retinoids: Biology, Chemistry, and Medicine,
2
nd Edition
, Raven Press, Ltd., New York, 1994, Chapter 8). Thus at least three distinct means are used to achieve HRE diversity: 1) binding site specificity for a particular half-site; 2) nucleotide spacing between the two half-sites; and 3) the orientation of the half-sites to one another.
In insect systems, a pulse of the steroid hormone ecdysone triggers metamorphosis in
Drosophila melanogaster
showing genomic effects, such as chromosomal puffing, within minutes of hormone addition. Mediating this response in insects is the functional ecdysone receptor, a heterodimer of the ecdysone receptor (EcR) and the product of the ultraspiracle gene (USP) (Yao et al. (1993)
Nature
366:476-479; and Yao et al. (1992)
Cell
71:63-72). Responsiveness to an insect ecdysteroid can be recreated in cultured mammalian cells by co-transfection of EcR, USP, an ecdysone responsive reporter, and treatment with ecdysone or the synthetic analog muristerone A.
The ability to manage the expression of genes introduced into mammalian cells and animals would further advance many areas of biology and medicine. For instance, methods that allow the intentional manipulation of gene expression would facilitate the analysis of genes whose gene products cannot be tolerated constitutively or at certain stages of development. Such methods would also be valuable for clinical applications such as gene therapy protocols, where the expression of a therapeutic gene must be regulated in accordance with the needs of the patient (Saez et al. (1997)
Current Opinion in Biotechnology
8:608-616). However, to be of broad benefit, gene regulation techniques must allow for rapid, robust and precise induction/repression of gene activity. Precise control of gene expression is an invaluable tool in studying, manipulating and controlling development and other physiological processes.
Early designs to direct gene expression in mammals were based on endogenous elements, such as cytokine response elements or heat-shock proteins. Due to a high level of basal expression in the uninduced state, and pleiotropic effects brought about by general inducing agents, these systems lack the specificity required to regulate genes in mammalian cells and organisms. (Saez et al., supra)
As another means for controlling gene expression in a mammalian system, an inducible tetracycline regulated system has been devised and utilized in transgenic mice, whereby gene activity is induced in the absence of the antibiotic and repressed in its presence (see, e.g, Gossen et al. (1992)
Proc. Natl. Acad. Sci.
89:5547-5551; Gossen et al.(1993)
TIBS
18:471-475; Furth et al. (1994)
Proc. Natl. Acad. Sci.
91:9302-9306; and Shockett et al. (1995)
Proc. Natl. Acad. Sci.
92:6522-6526). However, problems were noticed during the development of this system including toxicity of the tetracycline transactivator protein, the requirement for continuous treatment of tetracycline to repress expression, and the slow clearance of antibiotic from bone which interferes with quick and precise induction. While this system has been improved by the recent identification of a mutant tetracycline repressor which acts conversely as an inducible activator, the pharmacokinetics of tetracycline may hinder its use during development when a precise and efficient “on-off” switch is essential (Gossen et al. (1995)
Science
268:1766-1769).
Another approach to regulate gene expression relies on induction of protein dimerization, a method derived from studies on the mechanism of action of immunosuppressive agents (Spencer, DM, (1996)
Trends Genet,
12:181-187). Compounds such as FK506 and cyclosporin A (CsA) subdue the immune response by binding with high affinity to the immunophilins FKBP12 and cyclophilin (CyP), respectively. Using a synthetic homodimer of FK506 (called FK10102), a general strategy was devised to bring together any two peptides, by endowing them with the domain of FKBP12 to which FK506 binds. By chemically linking FK506 and CsA, a heterodimer molecule that can selectively connect two different immunophilin domains and their respectively attached peptides was also generated.
The resulting heterodimerizer, FKCsA, has been used to reconstitute a functional transcription factor by joining a GAL4 DNA-binding domain fused to FKBP12, and the transactivation moiety of VP16 bound to CyP. In cells expressing these chimeric proteins, the expression of a promoter containing GAL4 binding sites was strongly stimulated in the presence of this ‘chemical inducer of dimerization’ (CID) FKCsA.
Th
Evans Ronald M.
Saez Enrique
Clark Deborah J. R.
Foley & Lardner
Kaushal Sumesh
Reiter Stephen E.
The Salk Institute for Biological Studies
LandOfFree
Formulations useful for modulating expression of exogenous... does not yet have a rating. At this time, there are no reviews or comments for this patent.
If you have personal experience with Formulations useful for modulating expression of exogenous..., we encourage you to share that experience with our LandOfFree.com community. Your opinion is very important and Formulations useful for modulating expression of exogenous... will most certainly appreciate the feedback.
Profile ID: LFUS-PAI-O-2591480