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Methods and compositions for identifying morphogen analogs

a technology of morphogen analogs and compositions, applied in the field of screening and identifying substances, can solve the problems of affecting the effectiveness of drug effects, and presently encountered complications,

Inactive Publication Date: 2008-01-03
STRYKER CORP
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Benefits of technology

The approach enables the production of morphogen analogs that effectively induce tissue-specific morphogenesis, reduce production costs, and minimize undesirable side effects, making them suitable for treating metabolic bone diseases and other conditions.

Problems solved by technology

Certain complications, however, presently are encountered during the production, formulation and use in vivo of therapeutic macromolecules, such as morphogen proteins.
Providing such assurance can add significantly to the cost and technical difficulty of commercial production of biological macromolecules.
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.

Method used

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  • Methods and compositions for identifying morphogen analogs
  • Methods and compositions for identifying morphogen analogs
  • Methods and compositions for identifying morphogen analogs

Examples

Experimental program
Comparison scheme
Effect test

example 1

Effect of OP-1 on the Proliferation and Differentiation of C5.18 Cells

[0075] To characterize the biological effects of OP-1 on bone derived cell lines, OP-1 responsiveness was examined in C5.18 cells, spontaneously immortalized fetal rat calvaria cells well-known in the art and described, for example, in Grigoriadis et al. (1990) Developmental Biology 142:313-318 and in Von Schroeder et al. (1994) Teratology 50:54-62, the disclosures of which are herein incorporated by reference. C5.18 cells were plated in 12-well culture dishes (1×105 cell / well) in αMEM containing 15% fetal bovine serum. As described below, varying amounts of recombinant human OP-1 (Creative BioMolecules, Inc., Hopkinton, Mass.) were added to the culture media and the calvaria cells were incubated with the OP-1 containing medium for varying lengths of time as indicated below. OP-1 was prepared and formulated generally as earlier described in U.S. Pat. Nos. 5,258,494; 5,266,683; and 5,354,557, the disclosures of wh...

example 2

Effects of OP-1 on Type X Collagen Promoter Reporter Constructs

[0082] The above-described effect of OP-1 on the expression of type X collagen was of particular interest, as this phenotypic marker is generally understood to be specific for hypertrophic chondrocytes and thus of endochondral bone formation. The mouse type X collagen gene promoter was therefore employed as a model in order to examine the molecular mechanisms for OP-1 induction of chondrocyte phenotypic markers. The responsiveness of the type mouse X collagen gene promoter to OP-1 was therefore studied using the below described luciferase reporter gene deletion analyses:

[0083] The promoter region of the mouse type X collagen gene (nucleotides 1 to 1067, as designated by Elima et al. (1993) Biochem. J. 289:247-253, and by GenBank EMBL Data Bank: Accession #X67348; COLI0A1 gene; collagen alpha I type X) (also designated herein as nucleotides 1-1067 of SEQ. ID No. 1) was cloned according to well-known PCR (polymerase chai...

example 3

Mutation Analysis of the Transcription Activating Element

[0092] To characterize the respective roles of the AP-1 like (nucleotides 715-724 of SEQ. ID No. 1) and the A / T rich sequences (nucleotides 699-711 of SEQ. ID No. 1) (both of which are depicted within sequence “w” in FIG. 12) in the OP-1 driven induction of the collagen X promoter, mutations of the nucleotides 699-731 (SEQ. ID No. 1) upstream of the heterologous RSV promoter were analyzed (FIG. 12). Mutation of a portion of the AP-1 like sequence (nucleotides 715-724 of SEQ. ID No. 3) which changes the sequence (GAAT) to (TCCG) (depicted as sequence “M1” in FIG. 12) strongly suppressed the enhancer activity of this region and abolished the stimulatory effects of OP-1 (100 ng / ml) on this region. The above mutation also abolished the stimulatory effects of bFGF (10 ng / ml), which is capable of inducing the expression of the (699-731)x3-RSV construct in the presence of 1% serum (FIG. 12). In contrast, mutation of a portion of the...

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Abstract

Disclosed herein are methods and compositions for identifying morphogen analogs. The preferred methods and compositions relate to the discovery that morphogen upregulation of the mouse type X collagen promoter activity is mediated by a MEF-2 like sequence and requires an adjacent AP-1 sequence. Certain methods rest on the use of test cells comprising DNA defining a morphogen-responsive transcription activating element operatively associated with a reporter gene. Other methods rest on the use of DNAs for measuring morphogen-inducible DNA-binding. In certain preferred embodiments, the methods and DNAs involve an osteogenic protein 1 (OP-1) responsive transcription activating element. Substances that mediate interaction with and / or activate the OP-1 responsive transcription activating element are considered herein likely to be useful for reproducing in vivo effects of morphogens such as OP-1.

Description

REFERENCE TO RELATED APPLICATIONS [0001] The present application is a Continuation-in-Part application of co-pending U.S. Ser. No. 08 / 507,598, filed Jul. 26, 1995, the disclosure of which is herein incorporated by reference.FIELD OF THE INVENTION [0002] The present invention relates generally to methods and compositions for screening and identifying substances useful as morphogen analogs. In certain embodiments, the identified substances can be used to mimic a biological effect of morphogenic proteins such as osteogenic protein 1 (OP-1) on cellular gene expression and / or tissue-specific morphogenesis in mammals. BACKGROUND OF THE INVENTION [0003] Osteogenic Protein-1 of human origin (hOP-1), described in U.S. Pat. Nos. 5,011,691 and 5,266,683, and in Ozkaynak et al. (1990) EMBO J. 9: 2085-2093, recently has been appreciated to be competent to induce genuine tissue morphogenesis in mammals, including the endochondral morphogenesis of bone. It has further been appreciated that mouse O...

Claims

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Application Information

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Patent Type & Authority Applications(United States)
IPC IPC(8): C12N5/10C07H21/04C12N15/09A61K31/00A61K38/00A61K38/27A61K45/00A61P1/00A61P1/04A61P1/16A61P9/00A61P9/10A61P19/00A61P19/08A61P25/28A61P29/00A61P43/00C07K14/51C12Q1/68
CPCA61K38/00C07K14/51C07K14/4705A61P1/00A61P1/04A61P1/16A61P9/00A61P9/10A61P19/00A61P19/08A61P25/28A61P29/00A61P43/00
Inventor HARADA, SHUN-ICHIRODAN, GIDEON A.SAMPATH, KUBER T.
Owner STRYKER CORP
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