Chemistry: molecular biology and microbiology – Vector – per se
Reexamination Certificate
2001-11-21
2004-11-16
Wehbe′, Anne M. (Department: 1632)
Chemistry: molecular biology and microbiology
Vector, per se
C536S023100, C536S024100
Reexamination Certificate
active
06818446
ABSTRACT:
BACKGROUND OF THE INVENTION
1. Field of the Invention
The invention relates to compositions and methods for the assessment of mucin gene expression. The invention also relates to compositions and methods for the identification of compounds useful in the treatment of various medical conditions caused by mucin overproduction.
2. Description of the Related Art
Mucins are a family of high molecular weight glycoproteins secreted from epithelial cells at many body surfaces, including the eyes, pancreatic ducts, gallbladder, prostate and respiratory, gastrointestinal and reproductive tracts. Mucins are a major component of mucus, and are responsible for the viscoelastic properties of mucus, and serve a role in protecting and lubricating the epithelial surfaces. At least twelve mucin genes have been identified in humans.
In the airways, mucin proteins form a protective barrier on the airway epithelial cells, and interact with cilia to trap and clear pathogens (e.g., microorganisms), particulate matter, irritants and pollutants (e.g., tobacco smoke and sulfur dioxide). Mucus secretions in the airway are produced from two different secretory cell populations, the surface epithelial goblet cells and the mucous cells in the submucosal glands. At least eight mucin genes are expressed (at the mRNA level) in the upper and lower respiratory tracts. Of these, only the MUC5AC and MUC5B polypeptides have been conclusively demonstrated to be major components of human airway secretions (Hovenberg et al.,
Biochem. J.,
318(Pt. 1, Vol. 17):319-324 [1996]; Hovenberg at al.,
Glycoconjugate Jour.,
13(5):839-847 [1996]; Thornton et at.,
J. Biol. Chem.,
272(14):9561-9566 [1997]; and Wickstrom et al.,
Biochem. Jour.,
334(Pt. 3, Vol. 14):685-693 [1998]). MUC5B is also expressed in other tissues, including, for example, pancreas and gall bladder.
Diseases of Mucin Overproduction
Mucin production is upregulated in response to mucosal irritation. Most notably, bacterial infection of the airway epithelium is often accompanied by mucin overproduction. Some airway diseases are also characterized by mucus hypersecretion. Hypersecretion of mucus can overwhelm the ability of the cilia to function properly, and can result in various pathologies, such as airway mucus plugging and airflow obstruction. Mucus hypersecretion also contributes to chronic infection by shielding bacteria from endogenous and exogenous antibacterial agents. Mucus plugging and bacterial infections create a non-healing injury and can result in chronic influx of inflammatory cells which destroy gas exchange tissue. When severe, these effects result in respiratory function decline, and can be fatal.
Diseases which are characterized by mucin (and mucus) hypersecretion also frequently demonstrate goblet cell hyperplasia and submucosal gland hypertrophy. Such diseases include, for example, chronic bronchitis, bronchial pneumonia, cystic fibrosis, chronic asthma, emphysema, usual interstitial pneumonitis and other diseases (Basbaum et al.,
Am. Rev. Respir. Dis.,
144(3 Pt 2):S38-41 [1991]; Yanagihara et al.,
Am. J. Respir. Cell. Mol. Biol.,
24(1):66-73 [2001]; Rogers et al.,
Eur. Respir. J.,
7(9):1690-706 [1994]; and Kaliner et al.,
American Review of Respiratory Disease
134(3):612-21[1986]).
MUC5B mRNA and Genomic Structure
In order to better understand the molecular mechanism of mucin gene expression regulation in normal and disease states, it is necessary to elucidate the genomic structure of the mucin gene. MUC5B and three other mucin genes, MUC6, MUC2, and MUC5AC, have all been mapped to 11p15.5 on a single band of 400 kilobases, and their order has been determined to be: telomere-MUC6-MUC2-MUC5AC-MUC5B-centromere. The MUC5B genomic structure (i.e., exon identification, intron/exon boundaries and transcriptional start sites) and cDNA sequence are also partially known, albeit with some discrepancies in the published literature (Pigny et al.,
Genomics
38(3):340-352 [1996]; Desseyn et al.,
Jour. Biol. Chem.,
273(46):30157-30164 [1998]; Desseyn et al.,
Jour. Biol. Chem.,
272(27):16873-16883 [1997]; Desseyn et al.,
Jour. Biol. Chem.,
272(6):3168-3178 [1997]; Offner et al.,
Biochem. Biophy. Res. Comm.,
251(1):350-355 [1998]; and Keates et al.,
Biochem. J.,
324(Pt 1):295-303 [1997]).
The MUC5B gene is large and complex. The MUC5B exons and introns encompass approximately 39076 basepairs of genomic sequence, and the gene's cDNA is approximately 17079 basepairs in length. The gene is characterized by an unusually large central exon of 10,713 basepairs and 3,571 amino acids. The central exon contains multiple repeated motifs, including characteristic cysteine-rich subdomains, which are also found in other mucin genes. In addition to the large central exon, there are approximately 30 smaller exons upstream and another approximately 17 exons downstream of the central exon. In total, the MUC5B message is predicted to encode a 5683 amino acid polypeptide having a molecular weight of 590 kDa. However, as the mucin proteins are extensively glycosylated, the observed molecular weight is expected to be much greater. Conflicting descriptions of the gene's transcription start sites and identity of the first exon have been reported.
There exist published reports of the isolation and analysis of limited portions of the MUC5B 5′ promoter region. Van Seuningen et al. (
Biochem. J.,
348(Pt. 3):675-686 [2000]) describe an analysis of the MUC5B promoter region, which encompasses approximately 956 basepairs of genomic nucleotide sequence upstream of the transcription start site. Perrais et al. (
J. Biol. Chem.,
276(18):15386-15396 [2001]) describe an analysis of the MUC5B promoter region, which includes approximately 2044 basepairs of genomic nucleotide sequence upstream of the transcription start site. GenBank Accession Number AJ012453 describes approximately 2954 basepairs of MUC5B genomic sequence 5′ of the transcriptional start site.
There is a need to identify compounds capable of inhibiting the production of mucin proteins, and specifically, MUC5B protein. There is a need to provide therapies for reducing mucus (e.g., MUC5B) production in individuals suffering from airway diseases characterized by mucus hypersecretion, such as cystic fibrosis, chronic bronchitis, bronchial pneumonia and asthma. The object of the present invention is to provide novel compositions and methods that find use in the analysis of MUC5B gene expression. These compositions incorporate previously unreported MUC5B genomic sequences derived from the MUC5B gene 5′ promoter region, and the methods of the invention use these sequences. These novel compositions further comprise reporter genes in operable combination with the novel MUC5B gene 5′ promoter regions of the present invention. It is also an object of the present invention to provide methods for drug screening using the novel MUC5B promoter reporter constructs to identify compounds having the ability to downregulate MUC5B gene expression. The invention also provides transgenic animals suitable for use in screening assays to identify compounds capable of inhibiting mucin production. Compounds thus identified find use in the treatment of diseases characterized by mucin hypersecretion.
SUMMARY OF THE INVENTION
The present invention provides novel isolated nucleic acid molecules comprising promoter sequences regulating the transcription of the human MUC5B gene. These novel sequences are provided in SEQ ID NO: 31 and SEQ ID NO: 32. In a related embodiment, the invention also provides nucleic acid molecules wherein the promoter sequences of SEQ ID NO: 31 or SEQ ID NO: 32 are operably linked to a heterologous gene (i.e., a gene that is not naturally linked to the promoter sequences of SEQ ID NO: 31 or SEQ ID NO: 32).
In one embodiment, the combination of promoter sequence and heterologous gene reside within a vector. In some embo
Chen Yin
Wu Reen
Dreger Ginger R.
Heller Ehrman White & McAuliffe LLP
Li Q. Janice
The Regents of the University of California
Wehbe′ Anne M.
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