Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving nucleic acid
Reexamination Certificate
2001-05-15
2004-12-07
Kemmerer, Elizabeth (Department: 1647)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving nucleic acid
C435S069100, C435S320100, C435S325000, C536S023100
Reexamination Certificate
active
06828095
ABSTRACT:
FIELD OF THE INVENTION
The invention relates generally to the field of drug screening assays. More particularly, the invention relates to methods and compositions for identifying molecules that modulate expression of true tissue morphogenic proteins.
BACKGROUND OF THE INVENTION
A class of proteins recently has been identified, the members of which are true tissue morphogenic proteins. The members of this class of proteins are characterized as competent for inducing the developmental cascade of cellular and molecular events that culminate in the formation of new organ-specific tissue, including any vascular and connective tissue formation, as required by the naturally occurring tissue. Specifically, the morphogens are competent for inducing all of the following biological functions in a morphogenically permissive environment: (1) stimulating proliferation of progenitor cells; (2) stimulating differentiation of progenitor cells; (3) stimulating the proliferation of differentiated cells and (4) supporting the growth and maintenance of differentiated cells. For example, the morphogenic proteins can induce the full developmental cascade of bone tissue morphogenesis, including the migration and proliferation of mesenchymal cells, proliferation and differentiation of chondrocytes, cartilage matrix formation and calcification, vascular invasion, osteoblast proliferation, bone formation, bone remodeling, and hematopoietic bone marrow differentiation. These proteins also have been shown to induce true tissue morphogenesis of non-chondrogenic tissue, including dentin, liver, and nerve tissue.
A particularly useful tissue morphogenic protein is human OP-1 (Osteogenic Protein-1), described in U.S. Pat. No. 5,011,691; U.S. Pat. No. 5,266,683 and Ozkaynak et al. (1990)
EMBO J.
9: 2085-2093. Species homologues identified to date include, but are not limited to, mouse OP-1 (see U.S. Pat. No. 5,266,683) and the Drosophila homologue 60A, described in Wharton et al. (1991)
Proc. Nat. Acad. Sci. USA
88:9214-9218). Other closely related proteins include OP-2 (Ozkaynak (1992)
J. Biol. Chem.
2:25220-25227 and U.S. Pat. No. 5,266,683); BMP5, BMP6 (Celeste et al. (1991)
Proc. Natl. Acad. Sci.
8:9843-9847) and Vgr-1 (Lyons et al. (1989). These disclosures are incorporated herein by reference.
It previously has been contemplated that these tissue morphogens can be administered to an animal to regenerate lost or damaged tissue. Certain complications, however, presently are encountered during the production, formulation and use in vivo of therapeutic macromolecules, such as morphogen proteins. For example, such proteins are typically produced by fermentation or culture of suitable host cells. Any biological product produced from such host cells for use in humans presently must be shown to be essentially free of host cell contaminants, such as secreted or shed proteins, viral particles or degradation products thereof. Providing such assurance can add significantly to the cost and technical difficulty of commercial production of biological macromolecules. Furthermore, appropriate formulations must be developed for conferring commercially reasonable shelf life on the produced macromolecule, without significant loss of biological efficacy. An additional complicating factor arises when circumstances warrant an extended course of therapeutic treatment with the produced and formulated macromolecule: the treated mammal may develop an immunological response to the macromolecule, and any such response may interfere with effectiveness thereof. In extreme circumstances, treatment must be discontinued.
Alternatively, administering a molecule capable of modulating expression of the endogenous tissue morphogen is an effective means for providing morphogen to a site in vivo. For example, DNA sequences have been identified in the OP-1 gene promoter that resemble wt-1/Egr-1 consensus sequences, TLC binding sequences, FTZ binding sequences and steroid binding sequences (see WO 95/33831, the disclosure of which is incorporated herein by reference). Thus, molecules to which these regulatory sequences are responsive are likely modulators of the OP-1 gene and can influence its expression.
It is an object of this invention to provide compositions and methods of identifying compounds which can modulate expression of an endogenous tissue morphogen, particularly OP-1 and other members of the larger genus of true tissue morphogens. The compounds thus identified have utility both in vitro and in vivo. Useful compounds contemplated include at least those that are capable of stimulating transcription and/or translation of the OP-1 gene, as well as compounds capable of inhibiting transcription and/or translation of the OP-1 gene, via OP-1 non-coding DNA sequences resembling consensus sequences for Pax homeobox genes, in particular, the Pax 6 or Pax 2 genes.
These and other objects and features of the invention will be apparent from the description, drawings and claims which follow.
SUMMARY OF THE INVENTION
The invention features compositions and methods for screening candidate compounds for their ability to modulate the effective local or systemic levels of endogenous morphogen, particularly OP-1, in an organism. In one aspect, the method is practiced by: (I) incubating one or more candidate compounds with cells transfected with a DNA sequence encoding at least a portion of a morphogen non-coding DNA sequence that is responsive to a Pax homeobox gene and which is competent to act on and affect expression of a reporter gene with which it is operatively associated; (2) measuring the level of reporter gene expression in the transfected cell, and (3) comparing the level of reporter gene expressed in the presence of the candidate compound with the level of reporter gene expressed in the absence of the candidate compound. The level of an expressed reporter gene product in a given cell culture, or a change in that level resulting from exposure to one or more compound(s) indicates that the compound can also modulate the level of the morphogen normally associated with the non-coding sequence. Specifically, an increase in the level of reporter gene expression is indicative of a candidate compound's ability also to increase morphogen expression in vivo. Similarly, a decrease in the level of reporter gene expression is indicative of a candidate compound's ability also to decrease or otherwise interfere with morphogen expression in vivo. The above method is particularly useful for identifying compounds that are capable of influencing Pax mediated OP-1 gene expression.
The methods of the invention can therefore be used to identify compounds showing promise as therapeutics for various in vivo and ex vivo mammalian applications, as well as to identify compounds having numerous utilities. For example, compounds that modulate morphogen expression by stimulating Pax 2 or Pax 6 mediated transcription of a morphogen can be used in vivo to correct or alleviate a disease condition, to regenerate lost or damaged tissue, to induce cell proliferation and differentiation, and/or to maintain cell and tissue viability and/or a differentiated phenotype in vivo or ex vivo. The compounds also can be used to maintain the viability of, and the differentiated phenotype of, cells in culture. The various in vivo, ex vivo, and in vitro utilities and applications of the morphogenic proteins described herein are well documented in the art. See, for example, US 92/01968 (WO 94/03200), filed Mar. 11, 1992; US 92/07358 (WO 93/04692), filed August 28; PCT US 92/0743 (WO 93/05751), filed Aug. 28, 1992; US 93/07321 (WO 94/03200), filed Jul. 29, 1993; US 93/08808 (WO 94/06449), filed Sep. 16, 1993; US93/08885 (WO94/06420), filed Sep. 15, 1993, and U.S. Pat. No. 5,266,683.
In another aspect, the invention further provides vectors and cells useful for morphogen, particularly OP-1, therapy. In one embodiment, the invention features a vector having a reporter gene operatively associated with at least a portion of one or more OP-1 non-coding sequences responsive to Pax
Haimanti Dorai
Oppermann Herman
Sampath Kuber T.
Shepard Alyssa A.
Curis, Inc.
DeBerry Regina M.
Kemmerer Elizabeth
Ropes & Gray LLP
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