Osf2/Cbfa1 nucleic acids and methods of use therefor

Details Osf2/Cbfa1 nucleic acids and methods of use therefor Osf2/Cbfa1 nucleic acids and methods of use therefor

1998-05-29

2003-02-11

Nguyen, Dave T. (Department: 1632)

Chemistry: molecular biology and microbiology

Animal cell, per se ; composition thereof; process of...

C435S320100, C435S252300, C536S023500

Reexamination Certificate

active

06518063

ABSTRACT:

1.0 BACKGROUND OF THE INVENTION
1.1 Field of the Invention
The present invention relates generally to the field of molecular biology, and in particular, skeletogenesis. More particularly, certain embodiments concern nucleic acid segments comprising a gene that encodes a novel osteoblast-specific transcription factor, designated Osf2/Cbfa1. In certain embodiments, the invention concerns the use of these polynucleotide and polypeptide compositions to regulate osteoblast differentiation, and stimulate bone tissue formation, growth, repair and regeneration. Methods are also provided for identifying Osf2/Cbfa1 and Osf2/Cbfa1-related genes and polypeptides from biological samples, as well as methods and kits for identifying compounds that interact with Osf2/Cbfa1 polypeptides or polynucleotides, as well as compounds that alter or inhibit osteogenesis in an organism.
1.2 Description of Related Art
1.2.1 Bone Development
Regulatory factors involved in bone repair are known to include systemic hormones, cytokines, growth factors, and other molecules that regulate growth and differentiation. Various osteoinductive agents have been purified and shown to be polypeptide growth-factor-like molecules. These stimulatory factors are referred to as bone morphogenetic or morphogenic proteins (BMPs), and have also been termed osteogenic bone inductive proteins or osteogenic proteins (OPs). Several BMP- (or OP-) encoding genes have now been cloned and characterized; these have been assigned the common designations of BMP-1 through BMP-8. Although the BMP terminology is widely used, it may prove to be the case that there is an OP counterpart term for every individual BMP (Alper, 1994). Likewise, additional genes encoding OPs and BMPs are still being identified.
BMPs 2-8 are generally thought to be osteogenic, although BMP-1 is a more generalized morphogen (Shimell et al., 1991). BMP-3 is also called osteogenin (Luyten et al., 1989) and BMP-7 is also called OP-1 (Ozkaynak et al., 1990). BMPs are related to, or part of, the transforming growth factor-&bgr; (TGF-&bgr;) superfamily, and both TGF-&bgr;1 and TGF-&bgr;2 also regulate osteoblast function (Seitz et al., 1992). Several BMP (or OP) nucleotide sequences and polypeptides have been described in U.S. Pat. Nos. 4,795,804; 4,877,864; 4,968,590; and 5,108,753; including, specifically, BMP-1 disclosed in U.S. Pat. No. 5,108,922; BMP-2A (currently referred to as BMP-2) in U.S. Pat. Nos. 5,166,058 and 5,013,649; BMP-2B (currently referred to as BMP-4) disclosed in U.S. Pat. No. 5,013,649; BMP-3 in U.S. Pat. No. 5,116,738; BMP-5 in U.S. Pat. No. 5,106,748; BMP-6 in U.S. Pat. No. 5,187,076; BMP-7 in U.S. Pat. No. 5,108,753 and U.S. Pat. No. 5,141,905; and OP-1, COP-5 and COP-7 in U.S. Pat. No. 5,011,691 (each of which is specifically incorporated herein by reference in its entirety).
Other growth factors or hormones that have been reported to have the capacity to stimulate new bone formation include acidic fibroblast growth factor (Jingushi et al., 1990); estrogen (Boden et al., 1989); macrophage colony stimulating factor (Horowitz et al., 1989); and calcium regulatory agents such as parathyroid hormone (PTH) (Raisz and Kream, 1983). Skeletal development is a multi-step process. It includes patterning of skeletal elements, commitment of mesenchymal cells to chondrogenic and osteogenic lineages, followed by the terminal differentiation of precursor cells into three specialized cell types: the chondrocyte in cartilage, the osteoblast and osteoclast in bone. Many genes encoding either growth factors or transcription factors were shown through genetic studies in mice to control patterning of skeletal elements (Luo et al.; 1996a, 1996b). These genetic analyses showed also that mutations in these genes do not severely affect the differentiation of the skeleton specific cell types suggesting that patterning and cell differentiation in the skeleton are achieved through different genetic pathways. Consistent with this hypothesis, genes such as PTHrP and c-fos were shown to control chondrocyte and osteoclast differentiation respectively, without affecting skeletal patterning (Karaplis et al., 1994; Wang et al., 1992; Johnson et al., 1992). Little is known, however, about the molecular determinants specifically responsible for controlling osteogenesis, and in particular, osteoblast differentiation.
Analysis of Osf2, the osteoblast nuclear activity polypeptide that binds to OSE2, showed that it is immunologically related to the Cbfa transcription factors (Geoffroy et al., 1995; Merriman et al., 1995). The Cbfa proteins are the mouse homologues of Runt, a Drosophila pair-rule gene product required for neurogenesis and sexual differentiation (Gergen and Wieschaus, 1985; Kania et al., 1990). Runt and the Cbfa proteins have a high degree of homology in their DNA-binding domain, a 128-amino-acid long motif called the runt domain (Kagoshima et al., 1993). The mouse genome contains three known runt homologues encoding numerous isoforms with well-characterized expression patterns (Ogawa et al., 1993a; Bae et al., 1992; Wijmenga et al., 1995; Simeone et al., 1995). None of the described Cbfa transcripts has been shown to be expressed exclusively or predominantly in bone, suggesting that still unknown member(s) of the Cbfa family control osteoblast-specific expression of Osteocalcin. This prompted the search for such a novel member or members.
1.2.2 Transcription Factors
The control of all biological processes results from a balance between various positive and negative-acting factors which interact with DNA regulatory elements and with each other. These protein factors play a critical role in controlling the expression of proteins, and thus are critical to both normal and pathological processes. Understanding these protein factors and how they modulate gene expression is key to strategies for the development of agents to control disease initiation and progression.
Gene-specific transcription factors provide a promising class of targets for novel therapeutics directed to these and other human diseases for the following reasons. One, transcription factors offer substantial diversity. Over 300 gene-specific transcription factors have been described, and the human genome may encode as many as 3000. Hence, they provide as plentiful a target source as cell-surface receptors. Two, transcription factors offer substantial specificity. Each and every factor offers unique molecular surfaces to target. Three, transcription factors are known to be involved in human disease. For example, many tumors are associated with the activation of a specific oncogene. A third of known proto-oncogenes and three fourths of all anti-oncogenes are transcription factors.
Transcription factors are capable of sequence-specific interaction with a portion of a gene or gene regulatory region. The interaction may be directed sequence-specific binding where the transcription factor directly contacts the nucleic acid or indirect sequence-specific binding mediated or facilitated by other auxiliary proteins where the transcription factor is tethered to the nucleic acid by a direct nucleic acid binding protein. In addition, some transcription factor demonstrate induced or synergistic binding. A broad range of transcription factor-nucleic acid complexes provide useful targets. The gene and/or transcription factor may be derived from a host or from an infectious or parasitic organism. As examples, a host may be immunomodulated (e.g., by controlling inflammation or hypersensitivity) by modulating the DNA binding of a transcription factor involved in immune cell activation; or vital, bacterial, or other microbial disease progression may be inhibited by disrupting the DNA binding of a host, vital or other microbial transcription factor involved in vital or other microbial gene transcription.
1.3 Deficiencies in the Prior Art
What is lacking in the prior art, inter alia, are polynucleotide compositions that encode polypeptides that possess osteoblast-specific transcription factor activity. Also lacking are methods of re

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