Chemistry: molecular biology and microbiology – Micro-organism – tissue cell culture or enzyme using process... – Preparing compound containing saccharide radical
Reexamination Certificate
1998-09-18
2003-06-03
Nguyen, Dave Trong (Department: 1633)
Chemistry: molecular biology and microbiology
Micro-organism, tissue cell culture or enzyme using process...
Preparing compound containing saccharide radical
C435S069100, C435S320100, C435S325000, C435S455000, C536S023100, C536S024100, C536S024300, C536S025300, C536S024330
Reexamination Certificate
active
06573073
ABSTRACT:
Cystic fibrosis (CF) is one of the most common serious inherited diseases amongst Caucasians. One in 20-25 people harbour a mutation in the CF gene. As CF is an autosomal recessive syndrome this means that around 1 in 2,000 live births are affected. CF affects many organs including sweat glands (cystic fibrosis sufferers have salty sweat), the gut and the pancreas (85% of CF patients are pancreatic insufficient and require enzyme supplements in the diet). However, it is the effects on the lung which are most commonly life-threatening and lead to premature death. Specifically, cystic fibrosis patients accumulate mucus in the airways which is only relieved by regular physiotherapy. This mucus serves as a substrate for bacterial infection resulting in lung damage. More recently it has been shown that the cystic fibrosis lung milieu inactivates naturally occurring anti-microbial defences. The gene for cystic fibrosis was identified and sequenced in 1989 and subsequent studies showed that the gene product is a cAMP-activated chloride channel in the membrane of specialised epithelial cells.
Cystic fibrosis is an ideal model disease for gene therapy. First, it is a single gene disorder. Second, the most important target cells, within the airway epithelium, are accessible by non-invasive techniques. Third, at least one function of the CF gene product is known, allowing ready determination of whether the gene has been delivered and expressed in a functional form.
The cystic fibrosis transmembrane conductance regulator (CFTR) gene shows a tightly regulated pattern of temporal and spatial expression. Very little is known about the genetic elements and transcription factors that regulate CFTR expression. The basal promoter of the CFTR gene has been analysed in some detail though the data are somewhat inconsistent (Chou, J.-L. et al (1991) J. Biol. Chem. 266: 24471-24476; Yoshimura, K. et al (1991) Nucleic Acids Res. 19: 5417-5423; Yoshimura, K. et al (1991) J. Biol. Chem. 266: 9140-9144; Koh, J. et al (1993) J. Biol. Chem, 268: 15912-15921). The minimal promoter sequence found between −226 and +98 bp with respect to the major transcription site is sufficient to drive low levels of expression of a reporter gene
There is little data on the control of cell specificity of CFTR expression though there is ample evidence for such regulation. The human CFTR gene is expressed at significant levels mainly in the epithelia lining the pancreas, intestine, bile ducts, mate genital ducts and in certain regions of the airway epithelium including the inferior turbinate of the nose, the trachea and the serous portion of submucosal glands. There is IS evidence that expression of the CFTR gene may be hormonally regulated in epithelia within the reproductive system. Some degree of cell type specific control has been inferred for uncharacterised elements within the immediate 5′ untranslated region. DNAse I hypersensitive sites (DHS) are often associated with regulatory elements. A number of these sites that show some degree of correlation with CFTR expression have been observed between −3,000 bp relative to the transcription start site and +100 bp into intron 1. However these sites have only been examined in a few long-term cell lines that either do or do not express CFTR mRNA and protein and hence may not adequately reflect cell specific regulation of expression of the CFTR gene in vivo.
Since the expression control elements of the CFTR gene had not been well defined the inventor screened a larger region of genomic DNA than had been analysed previously in an attempt to identify these elements. The chromatin structure of 120 kb of genomic DNA 5′ to the CFTR gene was analysed in a number of CFTR expressing and non-expressing cell types, including primary genital duct epithelial cells in addition to long term cell lines. The inventor identified DNAse I hypersensitive sites within this region by screening with probes isolated from cosmid and phage clones. Novel DNAse I hypersensitive sites were observed at −79.5 kb and −20.5 kb 5′ to the ATG translation start codon of the CFTR coding sequence. Neither of these sites showed strong correlation with CFTR expression in the cell types investigated. Although they may play an important role in the complex series of events involved in the regulation of CFTR transcription, these data do not support the existence of cell specific control elements at these sites.
A Novel DNAse I Hypersensitive Site in Intron 1
More recently, the inventor identified a DNAse I hypersensitive site 10 kb downstream of the 3′ end of exon 1 of the CFTR gene. The presence of this site correlated well, quantitatively and qualitatively, with the levels of expression of the CFTR gene in both long term cell lines and primary genital duct epithelial cells. This is the first intronic regulatory element to be reported for the CFTR gene. Its location may, in part, explain the failure of several groups to elucidate the elements involved in regulating control of expression of CFTR, since previous analyses of the CFTR promoter region have been restricted to sequences 5′ to and including the first exon. These findings are described in Smith, A. N. et al (1996) J. Biol. Chem. 271: 9947-9954.
FIG. 1A
shows the location of the intron 1 DNAse I hypersensitive site and the relative positions of the XB5.0, H4.0 and EB1.7 subfragments of the cW44 cosmid that were used as probes to detect this site. Increasing amounts of DNAse I revealed a hypersensitive site within this 22 kb fragment yielding a major product of 8 kb. The hypersensitive site was located approximately 10 kb 3′ to the end of exon 1 of the CFTR gene. Based on CF Genetic Analysis Consortium nomenclature this site has been called 181+10 kb, where 181 refers to the last base in exon 1. This hypersensitive site has the potential to contain cell type expression control elements as it is seen only in cell lines that transcribe CFTR mRNA. As shown in Table 1 below, the relative degree of hypersensitivity of the site correlated with the relative levels of endogenous expression of CFTR. The high expressing colon carcinoma cell lines Caco2 and HT29 show the site most strongly; the pancreatic adenocarcinoma cell line Capan that expresses low levels of CFTR mRNA shows the hypersensitive site weakly and the breast carcinoma epithelial cell line MCF7 and the lymphoblastoid cell line 37566 do not show this site. Most importantly, cultured human fetal epididymis and vas deferens epithelial cells that express CFTR in vitro show the hypersensitive site.
The 1 kb of DNA that spans the hypersensitive site has been cloned and sequenced and a partial restriction map of 850 bp flanking this region is shown in FIG.
1
B. The nucleotide sequence has been submitted to the GenBank/EMBL Data Bank with accession number U47863.
Electrophoretic Mobility Shift Assays (EMSA) AND DNAse I Footprinting
Overlapping fragments of approximately 200 bp, 3/4 (TSR3-TSR4), 5/6 (TSR5-TSR6), and 7/8 (TSR7-TSR8) from within the 850 bp fragment of intron 1 were generated by PCR (see FIG.
1
B and Table 2). No specific gel mobility shifts were observed with fragments 3/4 or 5/6. However several proteins bound to fragment 7/8. Two gel mobility shift bands were generated by nuclear extracts from all cell lines tested, irrespective of whether they transcribe CFTR. At least one other protein complex was seen in longterm cell lines transcribing CFTR, Caco2 and HT29. Primary epididymis and primary vas deferens nuclear extracts also caused gel mobility shifts of fragment 7/8 and the formation of a complex of at least 3 components. These protein DNA complexes were specifically competed by excess cold fragment 7/8 but not by the 5/6 fragment.
Further mapping of the location of the DNA protein complexes detected by EMSA was achieved by competition with subfragments of the 7/8 element. Subsequent DNAse I footprinting of the 7/8 fragment following binding of nuclear extracts from the MCF7 and HPAF cells lines that do not transcribe CFT
Isis Innovation Limited
Nguyen Dave Trong
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