WO2000004142A1 - Mucines - Google Patents

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WO2000004142A1
WO2000004142A1 PCT/AU1999/000579 AU9900579W WO0004142A1 WO 2000004142 A1 WO2000004142 A1 WO 2000004142A1 AU 9900579 W AU9900579 W AU 9900579W WO 0004142 A1 WO0004142 A1 WO 0004142A1
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Prior art keywords
muc
nucleic acid
isolated
seq
gene
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PCT/AU1999/000579
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English (en)
Inventor
Stephanie Jane Williams
Toni Marie Antalis
Michael Andrew Mcguckin
David Charles Gotley
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The Council Of The Queensland Institute Of Medical Research
The Corporation Of The Trustees Of The Order Of The Sisters Of Mercy In Queensland
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Application filed by The Council Of The Queensland Institute Of Medical Research, The Corporation Of The Trustees Of The Order Of The Sisters Of Mercy In Queensland filed Critical The Council Of The Queensland Institute Of Medical Research
Priority to CA002333853A priority Critical patent/CA2333853A1/fr
Priority to AU48887/99A priority patent/AU4888799A/en
Publication of WO2000004142A1 publication Critical patent/WO2000004142A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • C07K14/4701—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals not used
    • C07K14/4727—Mucins, e.g. human intestinal mucin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy

Definitions

  • TITLE "MUCINS" FIELD OF THE INVENTION relates generally to nucleic acids corresponding to mammalian Mucin genes, and to polypeptides encoded thereby. More particularly, the present invention provides isolated nucleic acids which correspond to Mucin regulatory genes that are predominantly expressed in the colon. These Mucin genes are associated with disease conditions including colorectal cancer, breast cancer, cystic fibrosis, respiratory diseases, inflammatory bowel disease, ulcerative colitis and Crohn's disease and/or any other conditions associated with aberrant Mucin expression, altered properties of mucus or epithelial inflammatory processes involving Mucins. In particular, the present invention provides methods for the diagnosis and therapy of the abovementioned disease conditions. BACKGROUND OF THE INVENTION
  • the epithelial mucins are a family of secreted and cell surface glycoproteins expressed by epithelial tissues. They are characterised by a central polymorphic tandem repeat structure, which comprises most of the protein backbone, and a large number of O-linked carbohydrate side chains (Gum etal., 1995, Biochem. Soc. Trans. 23795). The complex structure and large size of these molecules makes it difficult to characterise them using classical biochemical techniques.
  • the genes are also difficult to clone because of their large size and the presence of GC-rich tandem repeats.
  • Ten mucin genes have been identified; MUC3, MUC4, MUC5AC, MUC5B, MUC6 and MUC8 have been partially cloned and full-length cDNA clones are available for MUC1 , MUC2, MUC7 and MUC9.
  • Mucins are known to contribute to pathology in a number of epithelial diseases including cystic fibrosis (CF), inflammatory bowel disease (IBD) and adenocarcinomas.
  • Gastrointestinal mucins which have been described to date include: the transmembrane mucins MUC1 and MUC4; the gel-forming mucins MUC2, MUC5AC and MUC6; and MUC3 which has an unclear structure and function.
  • Mucin genes or isolated nucleic acids corresponding thereto will be expressed in italicized form as MUC.
  • Mucin polypeptides will be expressed as MUC.
  • Immunohistochemical staining and Western blotting analysis with mature MUC1 -specific antibodies revealed that MUC1 became ectopically expressed in colorectal tumours and levels were significantly higher in primary tumours of patients with metastases.
  • Experimentally increased expression of gel-forming mucins resulted in increased metastasis in colon cancer cells in xenograft metastasis models (Ho et al., 1995, Int. J. Oncol. 7 913).
  • MUC3 is located on human chromosome 7q22, or an equivalent location on other mammalian chromosomes, and is primarily expressed under normal conditions in the small intestine (Shekels et al., 1998, Biochem J. 330 1301).
  • the Mucins constitute an incomplete family of genes and gene products implicated in a variety of disease conditions.
  • the present inventors have identified novel Mucin genes located on human chromosome 7q22, and isolated novel nucleic acids corresponding thereto.
  • the present inventors have found that these novel Mucin genes are predominantly expressed in the colon, and may be involved in cancer of the large bowel, cystic fibrosis, breast cancer, inflammatory bowel disease, ulcerative colitis respiratory diseases and Crohn's disease and/or any other conditions associated with aberrant Mucin expression, altered properties of mucus or epithelial inflammatory processes involving Mucins.
  • the present invention is broadly directed to an isolated MUC nucleic acid which corresponds to a MUC gene located on mammalian chromosome 7q22, or on a mammalian chromosome structurally or functionally equivalent thereto, which MUC gene is normally predominantly expressed in the colon.
  • the MUC gene of the present invention is MUC11. Accordingly, "a MUC11 nucleic acid” means an isolated nucleic acid of the invention which corresponds to the MUC11 gene.
  • the isolated MUC11 nucleic acid comprises a nucleotide sequence encoding an amino acid sequence which comprises SGLSEESTTSHSSPGSTHTTLSPASTTT (SEQ ID NO: 1).
  • the isolated MUC11 nucleic acid comprises a nucleotide sequence encoding the amino acid sequence according to SEQ ID NO:3. Even more preferably, the isolated MUC11 nucleic acid comprises a nucleotide sequence according to SEQ ID NO: 2.
  • the MUC gene of the present invention is MUC12.
  • a MUC12 nucleic acid means an isolated nucleic acid of the invention which corresponds to the MUC12 gene.
  • the isolated MUC12 nucleic acid comprises a nucleotide sequence encoding an amino acid sequence which comprises SGLSQESTTFHSSPGSTETTLAPASTTT (SEQ ID NO: 4).
  • the isolated MUC12 nucleic acid comprises a nucleotide sequence encoding the amino acid sequence according to SEQ ID NO:6.
  • the isolated MUC12 nucleic acid comprises a nucleotide sequence according to SEQ ID NO: 5.
  • the present invention resides in an isolated MUC polypeptide.
  • the isolated MUC polypeptide has an amino acid sequence according to SEQ ID NO: 3, hereinafter referred to as a "MUC11 polypeptide”.
  • the isolated MUC polypeptide has an amino acid sequence according to SEQ ID NO:6, hereinafter referred to as a "MUC12 polypeptide".
  • the present invention resides in an antibody specific for a MUC polypeptide (hereinafter referred to as an anti-MUC antibody).
  • the anti-MUC antibody is selected from the group consisting of:- (i) an anti-MUC11 IgM monoclonal antibody hereinafter referred to as M11.9; and (ii) an anti-MUC12 IgM monoclonal antibody hereinafter referred to as M 12.15.
  • the present invention resides in methods of detecting a MUC gene, a MUC gene transcript or a MUC polypeptide.
  • the fifth aspect extends to methods for detecting a polymorphism, deletion, mutation, truncation or expansion in a MUC gene, a MUC gene transcript or a MUC polypeptide, or detecting a level of expression thereof.
  • One embodiment of the fifth aspect is directed to use of an isolated MUC nucleic acid to determine whether a mammal has a disease condition, or a predisposition thereto.
  • Another embodiment is directed to use of an isolated MUC nucleic acid to determine whether a mammal has a disease condition, or a predisposition thereto.
  • Another embodiment is directed to use of an isolated
  • MUC polypeptide to determine whether a mammal has a disease condition, or a predisposition thereto.
  • the present invention provides a method of gene therapy of a disease condition in a mammal, said method including administering to said mammal a gene therapy construct which includes an isolated MUC nucleic acid as hereinbefore defined, to thereby alleviate one or more symptoms of said disease condition in said mammal.
  • the present invention provides a method of treating a disease condition in a mammal, said method comprising the step of administering to said mammal a pharmaceutically effective amount of a MUC polypeptide or an anti-MUC antibody.
  • the present invention resides in a pharmaceutical composition
  • a pharmaceutical composition comprising a MUC polypeptide or anti-MUC antibody, together with a pharmaceutically acceptable carrier and/or diluent.
  • the mammal is a human.
  • the "disease condition" is associated with aberrant Mucin expression, altered properties of mucus or epithelial inflammatory processes involving Mucins.
  • the disease condition is selected from the group consisting of colorectal cancer (CRC), cystic fibrosis (CF), inflammatory bowel disease (IBD), breast cancer (BC), Crohn's disease, ulcerative colitis, asthma and chronic bronchitis.
  • CRC colorectal cancer
  • CF cystic fibrosis
  • IBD inflammatory bowel disease
  • BC breast cancer
  • Crohn's disease ulcerative colitis
  • asthma chronic bronchitis
  • the disease condition is selected from the group consisting of colorectal cancer (CRC), cystic fibrosis (CF), inflammatory bowel disease (IBD) and breast cancer (BC).
  • CRC colorectal cancer
  • CF cystic fibrosis
  • IBD inflammatory bowel disease
  • BC breast cancer
  • FIG. 1(A) Autoradiograph of a differential display gel showing amplified products from RNA isolated from matched normal colon (N) and primary colorectal tumor (P) tissues. Differentially expressed bands dd29 (MUC12) and dd34 (MUCH) are arrowed.
  • FIG. 1(B) Northern blot analysis of total RNA from patient 101 hybridized with the dd29 probe to detect a MUC12 gene transcript (mRNA). Signal corresponding to 18S ribosomal RNA is shown as a loading control.
  • FIG. 1(D) Multiplex semi-quantitative RT-PCR showing amplification of MUC12 mRNA transcripts from matched normal colonic mucosa and primary tumor # 40, normal mucosa from patient
  • FIG. 1(E) Multiplex semi-quantitative RT-PCR showing amplification of MUC11 mRNA transcripts in matched normal colonic mucosa and primary tumors of patients # 40, 164, and 97 and six colorectal cancer cell lines. Amplification of ⁇ 2 -microglobulin ( ⁇ 2 -MG) is included as a measure of total RNA.
  • FIG. 1(F) Multiplex semi-quantitative RT-PCR showing amplification of MUC12 mRNA transcripts from matched normal colonic mucosa and primary tumors # 346, 84, 128, 97 and 316 and from five unpaired Dukes' stage D tumors (M) # 93, 361 , 107, 357 and 367.
  • FIG. 1(G) Multiplex semi-quantitative RT-PCR showing MUC11 mRNA transcripts in matched normal colonic mucosa and primary tumors of patients # 110, 346, 84, 128, and 348 and from five unpaired Dukes' stage D tumors (M) # 93, 107, 361 , 367 and 357.
  • Amplification of ⁇ 2 -microglobulin ( ⁇ 2 -MG) is included as a measure of total RNA.
  • Ma denotes molecular size markers in FIG 1 D-G.
  • FIG. 2 Predicted amino acid sequence of MUC12. Numbering of amino acids is given on the right. The consensus sequence of the degenerate tandem repeat structure is shown at the top.
  • FIG. 3 Amino acid sequence alignment of the carboxyl termini of MUC12, hMUC3 (amino acids 1-366), mMuc3 (Shekels et al.,
  • FIG. 4 Predicted amino acid sequence of MUCH showing the degenerate tandem repeat structure. The consensus sequence is shown at the top and amino acids not consistent with this sequence are shown in bold. Hyphens indicate gaps placed in order to optimize the amino acid alignment. A potential N-glycosylation site is shaded.
  • FIG. 5 mRNA tissue distribution of the 7q22 mucin gene family. Only those tissues showing a positive signal by Northern blot analysis are represented in the histogram. Sixteen tissues of neural origin, heart, aorta, skeletal muscle, bladder, stomach, testis, ovary, spleen, pituitary gland, adrenal gland, thyroid gland, salivary gland and mammary gland were negative for mucin mRNA expression. Expression was quantified by densitometry and is shown as a proportion of the tissue showing highest expression.
  • FIG. 6 Domain organization of the C-termini of human MUC12, hMUC3, the rodent Muc3 mucins and the rat and human MUC4 mucins. The relative size of domains is accurate except that the N-glycosylated domain adjacent to the mucin domain in MUC4 is shown at approximately one fifth of its actual size. Only the beginning of the large mucin domains are shown.
  • FIG. 7 Alignment of the first extracellular EGF-like domain of MUC12 with human EGF-like growth factors. Dark shading highlights identical amino acids and light shading indicates conservative amino acid substitutions.
  • FIG. 8 Schematic representation of MUC 11 cloning (A) and MUC 12 cloning (B).
  • FIG. 9 Normal colonic expression patterns of MUC11 (A, B) and
  • FIG. 10 Expression of MUC11 and MUC12 mRNA in normal colon as detected by RT-PCR.
  • Cytokeratin 20, (CK20) a colonic epithelial marker, was employed as a loading control.
  • 'RC denotes right colon
  • TC the transverse colon
  • 'LC the left colon
  • 'CA refers to the caecum
  • 'R' denotes the rectum.
  • FIG. 11 Expression of MUC11 and MUC12 mRNA in CRC cell lines as detected by RT-PCR.
  • the loading control is ⁇ 2 -microglobulin (B2MG) and 'M' denotes the molecular weight marker.
  • FIG. 12 Expression of MUC11 and MUC12 mRNA in IBD as detected by RT-PCR.
  • Cytokeratin 20 (CK20) a colonic epithelial marker, was employed as a loading control.
  • 'N' denotes tissues which appear macroscopically normal and 'D' refers to tissues reported to have IBD.
  • 'CA refers to the caecum, 'CO' the colon, 'LC the left colon, 'TC the transverse colon, 'RS' the recto- sigmoid colon, 'SI' the small intestine, 'IL'denotes the ileum and
  • FIG. 13 Expression of MUC11 and MUC12 mRNA in BC as detected by
  • the loading control is ⁇ 2 microglobulin denoted by B2MG and the molecular weight marker is denoted by 'M'.
  • the positive control was normal colonic cDNA from patient 164.
  • FIG 14 Northern blot analysis of MUC11 expression in normal colon
  • N primary CRC
  • P primary CRC
  • the present invention is predicated in part on the identification of novel MUCH and MUC12 genes which are normally predominantly expressed in the colon.
  • the isolated MUC nucleic acids and MUC genes of the invention may be useful in treatment and diagnosis of disease conditions associated with aberrant Mucin expression, altered properties of mucus or epithelial inflammatory processes involving Mucins.
  • disease conditions include but are not limited to cancer of the large bowel (CRC), cystic fibrosis (CF), inflammatory bowel disease (IBD), respiratory diseases such as asthma and chronic bronchitis, breast cancer (BC), ulcerative colitis and Crohn's disease.
  • the present invention is particularly directed to cancers of the large bowel, which includes the colon, rectum and anal canal, such as CRC, although it extends to biochemically, physiologically and/or genetically related cancers in other parts of the gastrointestinal tract.
  • the MUC genes are, for example, down-regulated in CRC.
  • predominantly expressed is meant that a MUC gene transcript or MUC polypeptide encoded by said MUC gene is expressed in the colon at a level greater than in any other organ.
  • associated with is meant that the disease condition displays symptoms consistent with aberrant Mucin expression, altered properties of mucus or epithelial inflammation involving Mucins. The disease association may be merely correlative or may reflect a causative role of Mucins in the disease condition.
  • cancer is used in its broadest sense to include malignant tumours, carcinomas and sarcomas.
  • a MUC nucleic acid "corresponds to" a MUC gene by being an isolated nucleic acid derived from said MUC gene, or a portion thereof.
  • said gene has components including amino acid coding sequences and non-coding sequences.
  • Non-coding sequences include, for example, introns and regulatory sequences which include a promoter, translation initiation and termination sequences and a polyadenylation sequence, for example.
  • the isolated MUC nucleic acid may therefore correspond to some or all of the aforementioned components of the corresponding MUC gene.
  • MUC12 was formerly known as dd 29 or
  • MUC10 MUC10 ox "dd29"
  • MUC polypeptide is encoded by an isolated MUC nucleic acid or by a MUC gene as hereinbefore defined.
  • Isolated MUC nucleic acids of the invention may be in DNA (e.g. cDNA or genomic DNA), RNA (e.g. mRNA) or hybrid DNARNA form, eithre in double-stranded or single-stranded form.
  • DNA e.g. cDNA or genomic DNA
  • RNA e.g. mRNA
  • hybrid DNARNA form eithre in double-stranded or single-stranded form.
  • single- stranded MUC nucleic acids include nucleic acids having sequences complementary to the nucleotide sequences of SEQ ID NO:2 and SEQ ID NO:5.
  • the isolated MUC nucleic acid of the invention comprises a nucleotide sequence having at least 60% identity to the nucleotide sequence according to SEQ ID NO:2, or a nucleotide sequence capable of hybridizing thereto under at least low stringency conditions.
  • the isolated MUC nucleic acid of the invention comprises a nucleotide sequence having at least 60% identity to the nucleotide sequence according to SEQ ID NO:5, or a nucleotide sequence capable of hybridizing thereto under at least low stringency conditions.
  • the nucleotide sequence has at least 75% identity. More preferably, the nucleotide sequence has at least 90% sequence identity.
  • identity is used herein in its broadest sense to include the number of exact nucleotide or amino acid matches having regard to an appropriate alignment using a standard algorithm, such as but not limited to the Geneworks program (Intelligenetics).
  • BLAST family programs may also be useful (Altschul et al., 1997, Nucl. Acids Res. 25 3389, which is herein incorporated by reference).
  • a detailed discussion of sequence analysis can be found in Unit 19.3 of CURRENT PROTOCOLS IN MOLECULAR BIOLOGY, Eds Ausubel et al., (John Wiley & Sons), which is herein incorporated by reference.
  • the nucleotide sequence is capable of hybridizing under medium stringency conditions.
  • nucleotide sequence is capable of hybridizing under high stringency conditions
  • Reference herein to low stringency conditions includes and encompasses from at least about 1 % v/v to at least about 15% v/v formamide and from at least about 1 M to at least about 2 M salt for hybridisation at 42°C, and at least about 1 M to at least about 2 M salt for washing at 42°C.
  • Low stringency conditions also include 1 % Bovine Serum Albumin (BSA), 1 mM EDTA, 0.5 M NaHPO 4 (pH 7.2), 7% SDS for hybridization at 65°C, and (i) 2xSSC, 0.1 % SDS; or (ii) 0.5% BSA, 1 mM
  • BSA Bovine Serum Albumin
  • Medium stringency conditions include and encompass from at least about 16% v/v to at least about 30% v/v formamide and from at least about 0.5 M to at least about 0.9 M salt for hybridisation at 42°C, and at least about 0.5 M to at least about 0.9 M salt for washing at 42°C.
  • Medium stringency conditions also include 1 % Bovine Serum Albumin (BSA), 1 mM EDTA, 0.5 M NaHPO 4 (pH 7.2), 7% SDS for hybridization at 65°C, and (i) 2 x SSC, 0.1% SDS; or (ii) 0.5% BSA, 1 mM EDTA, 40 mM NaHPO 4 (pH 7.2), 5% SDS for washing at 42°C.
  • BSA Bovine Serum Albumin
  • High stringency includes and encompasses from at least about 31 % v/v to at least about 50% v/v formamide and from at least about 0.01 M to at least about 0.15 M salt for hybridisation at 42°C, and at least about 0.01 M to at least about 0.15 M salt for washing at 42°C. High stringency also includes 1% BSA, 1 mM EDTA, 0.5 M
  • NaHPO 4 (pH 7.2), 7% SDS for hybridization at 65°C, and (i) 0.2 x SSC, 0.1 % SDS; or (ii) 0.5% BSA, 1mM EDTA, 40 mM NaHPO 4 (pH 7.2), 1 % SDS for washing at a temperature in excess of 65°C.
  • T m of a duplex DNA decreases by 1 °C with every increase of 1% in the number of mismatched based pairs.
  • MUC genes and isolated MUC nucleic acids of the present invention are exemplified in relation to the human mammalian species, the present invention extends to orthologs in non-human mammals such as in primates, laboratory test animals (e.g. mice, rates, rabbits, guinea pigs, hamsters), companion animals (e.g. dogs, cats), livestock animals (e.g. sheep, pigs, horses, donkeys, cows) and captive wild animals (e.g. deer, fox).
  • laboratory test animals e.g. mice, rates, rabbits, guinea pigs, hamsters
  • companion animals e.g. dogs, cats
  • livestock animals e.g. sheep, pigs, horses, donkeys, cows
  • captive wild animals e.g. deer, fox
  • MUC homologs is used to encompass MUC orthologs, isolated nucleic acids which hybridize to MUC nucleic acids of the invention and isolated nucleic acids which display at least 60% sequence identity to isolated MUC nucleic acids.
  • MUC homologs encompass single or multiple nucleotide substitutions, deletions and/or additions to the isolated MUC nucleic acids of the invention, inclusive of mutants, fragments, parts, portions and segments of the nucleotide sequences of the invention.
  • the isolated MUC nucleic acids of the present invention and homologs thereof therefore include oligonucleotides, primers (such as for PCR), antisense sequences, molecules suitable for use in co-suppression and fusion nucleic acid molecules. Ribozymes are also contemplated by the present invention.
  • probes, primers and antisense sequences correspond to distinct portions of isolated MUC nucleic acids of the invention, in that they contain nucleotide sequences based on said distinct portions of an isolated MUC nucleic acid sequence.
  • Such probe and primer sequences may be based on a MUC sequence of the invention by being identical thereto, or by being degenerate with respect thereto.
  • oligonucleotides are nucleic acids which comprise a contiguous sequence of no more than seventy (70) nucleotides
  • polynucleotides are nucleic acids which comprise a contiguous sequence of more than seventy (70) nucleotides.
  • a “probe” may be an oligonucleotide or a polynucleotide, either double-stranded or single- stranded, for use in hybridization techniques such as Northern blotting, Southern blotting or in situ hybridization.
  • hybridization techniques such as Northern blotting, Southern blotting or in situ hybridization.
  • in situ hybridization also includes Fluorescence In Situ Hybridization (FISH), which is used for determining chromosomal localization.
  • FISH Fluorescence In Situ Hybridization
  • a "primer” is a nucleic acid (usually an oligonucleotide) capable of annealing to a nucleic acid template under appropriate conditions of ionic strength and temperature, which annealed primer can be extended in a template-dependent fashion by a suitable nucleic acid polymerase (for example Taqr polymerase or SequenaseTM). It will therefore be understood that primers of the invention may be useful for PCR, sequencing, RACE, primer extension and the like.
  • a suitable nucleic acid polymerase for example Taqr polymerase or SequenaseTM
  • isolated MUC nucleic acids, probes and primers may be modified such as by end-labeling with 32 P-ATP and T4 polynucleotide kinase or by random primed labeling with 32 P-dCTP and DNA polymerase. Biotinylation is also contemplated, as is modification with phosphorothiorates, fluorochromes, digoxigenin, enzymes and peptides, for example.
  • diagnostic methods may be employed which utilize isolated MUC nucleic acids of the present invention, or portions thereof such as probes and PCR primers. Also, diagnostic methods employing MUC polypeptides will be discussed in more detail hereinafter.
  • Diagnostic methods may include detection of MUC genes, transcripts and/or polypeptides in samples such as fecal specimens and/or in colonic biopsies, analysis of serum MUC levels in patients with epithelial diseases including cancers, breast tissue biopsy samples or in respiratory mucus samples from patients suffering from CF, asthma or chronic bronchitis.
  • the diagnostic methods of the present invention may therefore be applicable to determining whether an individual has a disease condition associated with aberrant Mucin expression, altered properties of mucus or epithelial inflammatory processes involving Mucins, or a predisposition to said disease.
  • predisposition refers to an increased probability that an individual will contract the disease.
  • the diagnostic methods may also indicate whether an individual actually suffers from the disease, assist in assessing the severity of disease, a prognosis of the likely course of disease and appropriate treatments for the disease.
  • the diagnostic methods of the invention may be useful whether or not the individual suffers from one or more symptoms of the disease.
  • the present invention therefore contemplates methods of detecting MUC genes and MUC gene transcripts (e.g. mRNA), such as involving hybridization techniques (for example, by Northern or Southern blotting or in situ hybridization) or polynucleotide sequence amplification techniques (for example RT-PCR). Such methods may detect:-
  • a polymorphism, deletion, mutation, expansion, and/or truncation in a MUC gene or MUC gene transcript (i) a polymorphism, deletion, mutation, expansion, and/or truncation in a MUC gene or MUC gene transcript; and (ii) a relative level of expression of a MUC gene transcript (an mRNA transcript derived from a MUC gene).
  • Such methods of detection facilitate determination of whether said MUC gene is aberrantly-expressed as an indication of a disease condition or a predisposition thereto. Also, MUC gene polymorphisms, deletions, mutations, truncations or deletions may be detected which indicate a disease condition or a predisposition thereto.
  • PCR is the preferred nucleic acid sequence amplification technique
  • polynucleotide sequence amplification techniques include rolling circle amplification (RCA) and strand displacement amplification (SDA).
  • RCA reference is made to WO97/19193 which is herein incorporated by reference.
  • SDA reference is made to U.S. Patent No. 5455166, which is herein incorporated by reference.
  • PCR includes within its scope RT-PCR and multiplex PCR as will be described in detail hereinafter. Such methods may be used for qualitative or semi-quantitative analysis.
  • PCR-RFLP Restriction Fragment Length Polymorphism
  • Northern, Southern and in situ hybridization methods involve formation of a hybrid nucleic acid comprising a MUC gene or mRNA transcript and a corresponding isolated MUC nucleic acid or portion thereof.
  • RNA isolation and Northern hybridization methods are described in detail herein, although the skilled person is also referred to Chapter 4 of CURRENT PROTOCOLS IN MOLECULAR BIOLOGY, Eds Ausubel et al., (John Wiley & Sons), which is herein incorporated by reference.
  • determining whether a MUC gene or MUC gene transcript includes a polymorphism, mutation, deletion, truncation and/or expansion can be performed using methods such as PCR-RFLP analysis, Single Strand Conformational Polymorhpism (SSCP) analysis and Denaturing Gradient Gel Electrophoresis (DGGE). These techniques have become well known in the art of mutation detection.
  • DGGE Single Strand Conformational Polymorhpism
  • DGGE Denaturing Gradient Gel Electrophoresis
  • a non-limiting example of DGGE is provided in Folde & Loskoot, 1994, Hum. Mut. 3 83, which is herein incorporated by reference.
  • a non-limiting example of specific allele detection by PCR-RFLP and SSCP is provided in Lappalainen et al., 1995, Genomics 27 274, which is herein incorporated by reference.
  • MUCH or MUC12 genes are associated with bowel cancers (CRC), CF, BC, IBD, chronic bronchitis, asthma, ulcerative colitis and/or Crohn's disease. These are examples of disease conditions associated with aberrant Mucin expression, altered properties of mucus or epithelial inflammatory processes involving Mucins.
  • the isolated MUC nucleic acids now provide a means for genetic screening of the abovementioned disease conditions in human and other mammalian species. Genetic screening may be conducted by determining full expression or full-length transcript production by Northern blot, cloning and sequencing of the MUC genes or identifying mutations by oligonucleotide hybridisation or by direct sequencing of PCR amplification products of the MUC genes.
  • the present invention extends to nucleic acid molecules having translation-terminating mutations leading to truncation mutants.
  • the detection of truncation mutants may be important for genetic analysis of people with, for example, cancer of the large bowel or with a propensity to develop large bowel cancer, determined on, for example, hereditary grounds.
  • Truncated MUC polypeptides may also be useful in developing therapeutic agents such as antagonists or for developing antibodies. Truncational mutants may be readily detected by a direct protein truncation test. In essence, DNA fragments including PCR amplification products or corresponding mRNA molecules are subjected to in vitro translation and optionally also transcription and the translation products assayed by, for example, SDS-PAGE or by differential antibody binding assays. This assay may also be employed to screen for agents capable of inducing truncation mutations or for agents acting as antagonists for truncation mutant-inducing agents.
  • MUC polypetides may be assayed by, for example, by antibody screening such as in an ELISA.
  • polypeptides which comprise an amino acid sequence having at least 60% identity to a MUC polypeptide amino acid sequence, preferably at least 75% identity thereto, or more preferably at least 90% identity thereto; and (ii) polypeptides encoded by MUC homologs.
  • MUC homologs Such polypeptides are hereinafter referred to as "MUC homologs”.
  • the MUC polypeptide homologs of the invention include amino acid substitution(s), deletion(s) and/or addition(s) to a MUC polypeptide sequence.
  • Particular examples include antigenic fragments and analogues useful in immunoassays and as therapeutic agents as well as other fragments carrying B cell and/or T cell linear or conformational epitopes.
  • Fusion polypeptides include the MUC polypeptide(s) together with fusion partners such as HIS 6 , glutathione-s-transferase (GST), thioredoxin (TR) and maltose binding protein (MBP). Fusion partners greatly assist recombinant synthetic polypeptide purification by virtue of each fusion partner affording affinity purification by a specific affinity matrix.
  • the fusion polypeptide also includes a protease-specific cleavage site, so that the fusion partner may be cleaved and removed following purification to leave a substantially unmodified MUC polypeptide.
  • fusion partners for purification of recombinant expressed polypeptides
  • purification systems such as the QIAexpressTM (HIS) 6 system, the Pharmacia GST purification system and the New England Biolabs MBP system.
  • epitope tags are well known in the art and include c-myc, influenza hemagglutinin and FLAG tags.
  • Green Fluorescent Protein is a well known fusion partner applicable to MUC polypeptides of the invention.
  • a particularly useful application of GFP fusion partners is in the visible identification of cells or tissues which express a GFP-MUC fusion polypeptide of the invention. Identification may be performed by flow cytometry or fluorescence microscopy, as are well known in the art.
  • MUC polypeptides and MUC homologs of the invention may be in recombinant form of may be chemically synthesized, as is well known in the art. Chemical synthesis is preferably suited to production of MUC peptides. As used herein, "peptides" have no more than fifty (50) contiguous amino acids.
  • MUC polypeptides are in recombinant form.
  • isolated MUC nucleic acids of the present invention may be ligated into an expression vector to form an expression construct capable of directing expression of said MUC nucleic acid in a prokaryotic cell (for example, E. coli) or in a eukaryotic cell (for example, yeast cells, fungal cells, insect cells, mammalian cells or plant cells).
  • a prokaryotic cell for example, E. coli
  • a eukaryotic cell for example, yeast cells, fungal cells, insect cells, mammalian cells or plant cells.
  • the expression vector comprises one or more regulatory elements which direct expression of the nucleic acid ligated in said expression construct.
  • regulatory sequences include promoters, enhancers, splice donor/acceptor sites, polyadenylation sequences, translation initiation (Kozak sequences) and translation termination signals.
  • Suitable promoters may be constitutive (for example, CMV- or SV40-derived promoters) or inducible (for example, Zn responsive metallothionein promoters) or repressible (fef-repressible promoters).
  • “Analogues" of the MUC polypeptides of the invention contemplated herein include, but are not limited to, modification to side chains, incorporating of unnatural amino acids and/or their derivatives during peptide, polypeptide or protein synthesis and the use of crosslinkers and other methods which impose conformational constraints on the proteinaceous molecule or their analogues. Such chemical analogues may be useful in providing stable means for diagnostic purposes or for producing agonists or antagonists or for producing stable molecules for use in natural product screening.
  • side chain modifications contemplated by the present invention include modifications of amino groups such as by reductive alkylation by reaction with an aldehyde followed by reduction with NaBH ⁇ amidination with methylacetimidate; acylation with acetic anhydride; carbamoylation of amino groups with cyanate; trinitrobenzylation of amino groups with 2, 4, 6-trinitrobenzene sulphonic acid (TNBS); acylation of amino groups with succinic anhydride and tetrahydrophthalic anhydride; and pyridoxylation of lysine with pyridoxal-5-phosphate followed by reduction with NaBH 4 .
  • modifications of amino groups such as by reductive alkylation by reaction with an aldehyde followed by reduction with NaBH ⁇ amidination with methylacetimidate; acylation with acetic anhydride; carbamoylation of amino groups with cyanate; trinitrobenzylation of amino groups with 2, 4, 6-trinitrobenzene sulphonic acid (TNBS); acylation of
  • the guanidine group of arginine residues may be modified by the formation of heterocyclic condensation products with reagents such as 2,3-butanedione, phenylglyoxal and glyoxal.
  • the carboxyl group may be modified by carbodiimide activation via O-acylisourea formation followed by subsequent derivitisation, for example, to a corresponding amide.
  • Sulphydryl groups may be modified by methods such as carboxymethylation with iodoacetic acid or iodoacetamide; performic acid oxidation to cysteic acid; formation of a mixed disulphides with other thiol compounds; reaction with maleimide, maleic anhydride or other substituted maleimide; formation of mercurial derivatives using 4-chloromercuribenzoate, 4-chloromercuriphenylsulphonic acid, phenylmercury chloride, 2- chloromercuri-4-nitrophenol and other mercurials; carbamoylation with cyanate at alkaline pH.
  • Tryptophan residues may be modified by, for example, oxidation with N-bromosuccinimide or alkylation of the indole ring with 2- hydroxy-5-nitrobenzyl bromide or sulphenyl halides.
  • Tyrosine residues on the other hand, may be altered by nitration with tetranitromethane to form a 3-nitrotyrosine derivative.
  • Modification of the imidazole ring of a histidine residue may be accomplished by alkylation with iodoacetic acid derivatives or N- carbethoxylation with diethylpyrocarbonate.
  • Examples of incorporating unnatural amino acids and derivatives during peptide synthesis include, but are not limited to, use of norleucine, 4-amino butyric acid, 4-amino-3-hydroxy-5-phenylpentanoic acid, 6-aminohexanoic acid, t-butylglycine, norvaline, phenylglycine, omithine, sarcosine, 4-amino-3-hydroxy-6-methylheptanoic acid, 2-thienyl alanine and/or D-isomers of amino acids.
  • peptides can be conformationally constrained by, for example, incorporation of C ⁇ and N ⁇ -methylamino acids, introduction of double bonds between C ⁇ and C p atoms of amino acids and the formation of cyclic peptides or analogues by introducing covalent bonds such as forming an amide bond between the N and C termini, between two side chains or between a side chain and the N or C terminus.
  • the present invention further contemplates chemical analogues of the polypeptides of the invention capable of acting as antagonists or agonists thereof, or which can act as functional analogues thereof.
  • Chemical analogues may not necessarily be derived from the polypeptides of the invention, but may share certain conformational similarities. Alternatively, chemical analogues may be specifically designed to mimic certain physiochemical properties of MUC poypeptides. Chemical analogues may be chemically synthesised or may be detected following, for example, natural product screening. Useful sources for screening for natural products include coral, reefs, sea beds, river beds, plants, microorganisms and aqua and antarctic environments.
  • Still another aspect of the present invention is directed to antibodies specific for MUC polypeptides and/or homologs thereof.
  • the anti-MUC antibody is M11.9. In another embodiment, the anti-MUC antibody is M12.15.
  • a detailed method of anti-MUC antibody preparation is provided hereinafter. In this regard, it will be understood that anti-MUC polypeptide antibodies may be produced by immunization with MUC polypeptides or MUC peptides.
  • anti-MUC antibody is an antibody specific for, or at least binds to, a
  • the anti-MUC antibodies of the present invention may be useful as therapeutic or diagnostic agents.
  • a MUC polypeptide or homolog can be used to screen for naturally occurring anti-MUC antibodies. These may occur, for example in some autoimmune diseases.
  • anti-MUC antibodies can be used to screen for MUC polypeptides.
  • Techniques for such assays are well known in the art and include, for example, sandwich assays and ELISA Knowledge of endogenous MUC polypeptide levels may be important for diagnosis of large bowel cancer or a predisposition to large bowel cancers or for monitoring certain therapeutic protocols. This knowledge may also be important in other epithelial cancers such as cancer of the breast.
  • Anti-MUC antibodies of the present invention may be monoclonal or polyclonal. Alternatively, fragments of antibodies may be used such as Fab fragments. Furthermore, the present invention extends to recombinant and synthetic antibodies and to antibody hybrids. A "synthetic antibody” is considered herein to include fragments and hybrids of antibodies. The antibodies of this aspect of the present invention are particularly useful for immunotherapy and may also be used as a diagnostic tool for assessing cancer development or cancer cell apoptosis or monitoring the program of a therapeutic mitum.
  • anti-MUC antibodies can be used to screen for endogenous MUC polypeptides.
  • the latter would be important, for example, as a means for screening for levels of the MUC polypeptide in a cell extract or other biological fluid or purifying the MUC polypeptide made by recombinant means from culture supernatant fluid.
  • Techniques for the assays contemplated herein are known in the art and include, for example, sandwich assays and ELISA.
  • second antibodies (monoclonal, polyclonal or fragments of antibodies or synthetic antibodies) directed to the first mentioned antibodies discussed above. Both the first and second antibodies may be used in detection assays or a first antibody may be used with a commercially available anti-immunoglobulin antibody.
  • An antibody as contemplated herein includes any antibody specific to any region of the MUC polypeptide.
  • Both polyclonal and monoclonal antibodies are obtainable by immunization with the enzyme or protein and either type is utilizable for immunoassays.
  • the methods of obtaining both types of sera are well known in the art.
  • Polyclonal sera are less preferred but are relatively easily prepared by injection of a suitable laboratory animal with an effective amount of a MUC polypeptide, or antigenic parts thereof, collecting serum from the animal, and isolating specific sera by any of the known immunoadsorbent techniques.
  • antibodies produced by this method are utilizable in virtually any type of immunoassay, they are generally less favoured because of the potential heterogeneity of the product.
  • the use of monoclonal antibodies in an immunoassay is particularly preferred because of the ability to produce them in large quantities and the homogeneity of the product.
  • the preparation of hybridoma cell lines for monoclonal antibody production derived by fusing an immortal cell line and lymphocytes sensitized against the immunogenic preparation can be done by techniques which are well known to those who are skilled in the art.
  • the present invention contemplates a method for detecting a MUC polypeptide in a protein extract obtained from a mammal, said method including the step of forming a complex between an anti-MUC antibody and a MUC polypeptide, and then detecting said complex.
  • MUC polypeptide may be determined in a number of ways such as by Western blotting and ELISA procedures.
  • a wide range of immunoassay techniques are available as can be seen by reference to U.S. Patent Nos. 4,016,043, 4,424,279 and 4,018,653. These include both single-site and two-site or "sandwich" assays of the non-competitive types, as well as in the traditional competitive binding assays. These assays also include direct binding of a labelled antibody to a target.
  • Sandwich assays are among the most useful and commonly used assays and are favoured for use in the present invention.
  • an unlabelled antibody is immobilized to a solid substrate and the sample to be tested brought into contact with the bound molecule.
  • a second antibody specific to the antigen is then added and incubated, allowing time sufficient for the formation of another complex of antibody-antigen-labelled antibody.
  • the protein extract might be a cell extract, tissue biopsy or possibly serum, saliva, mucosal secretions, lymph, tissue fluid and gastrointestinal fluid.
  • the extract is, therefore, generally a biological sample.
  • a first antibody having specificity for MUC or antigenic parts thereof is either covalently or passively bound to a solid surface.
  • the solid surface is typically glass or a polymer, the most commonly used polymers being cellulose, polyacrylamide, nylon, polystyrene, polyvinyl chloride or polypropylene.
  • the solid supports may be in the form of tubes, beads, discs of microplates, or any other surface suitable for conducting an immunoassay.
  • the binding processes are well- known in the art and generally consist of cross-linking covalently binding or physically adsorbing, the polymer-antibody complex is washed in preparation for the test sample.
  • An aliquot of the sample to be tested is then added to the solid phase complex and incubated for a period of time sufficient (e.g. 2- 40 minutes or overnight if more convenient) and under suitable conditions (e.g. from 4°C to 37°C) to allow binding of any subunit present in the antibody.
  • the solid phase complex is washed and dried and incubated with a second antibody which is specific for a portion of the antigen (i.e. MUC).
  • MUC a portion of the antigen
  • the second antibody is linked to a reporter molecule which is used to indicate the binding of the second antibody to MUC.
  • An alternative method involves immobilizing the target molecules in the biological sample and then exposing the immobilized target to specific antibody which may or may not be labelled with a reporter molecule.
  • reporter molecule a bound target may be detectable by direct labelling with the antibody.
  • a second labelled antibody specific to the first antibody is exposed to the target-first antibody complex to form a target- first antibody-second antibody tertiary complex.
  • the complex is detected by the signal emitted by the reporter molecule.
  • reporter molecule is meant a molecule which, by its chemical nature, provides an analytically identifiable signal which allows the detection of antigen-bound antibody. Detection may be either qualitative or quantitative.
  • the most commonly used reporter molecules in this type of assay are either enzymes, fluorochromes or radionuclide containing molecules (i.e. radioisotopes) and chemiluminescent molecules.
  • an enzyme is conjugated to the second antibody, such as via glutaraldehyde or periodate amongst other means.
  • glutaraldehyde or periodate amongst other means.
  • Commonly used enzymes include horseradish peroxidase, glucose oxidase, beta-galactosidase and alkaline phosphatase, amongst others.
  • the substrates to be used with the specific enzymes are generally chosen for the production, upon hydrolysis by the corresponding enzyme, of a detectable colour change. It is also possible to employ fluorogenic substrates, which yield a fluorescent product rather than the chromogenic substrates noted above.
  • the enzyme-labelled antibody is added to the first antibody-antigen complex, allowed to bind, and then the excess reagent is washed away. A solution containing the appropriate substrate is then added to the complex of antibody-antigen-antibody. The substrate will react with the enzyme linked to the second antibody, giving a qualitative visual signal, which may be further quantitated, usually spectrophotometrically, to give an indication of the amount of antigen which was present in the sample.
  • reporter molecule also extends to use of cell agglutination or inhibition of agglutination such as red blood cells on latex beads, and the like.
  • fluorescent compounds such as fluorescein and rhodamine
  • fluorescein and rhodamine may be chemically coupled to antibodies without altering their binding capacity.
  • the fluorochrome-labelled antibody When activated by illumination with light of a particular wavelength, the fluorochrome-labelled antibody adsorbs the light energy, inducing a state of excitability in the molecule, followed by emission of the light at a characteristic colour visually detectable with a light microscope.
  • the fluorescent labelled antibody is allowed to bind to the first antibody-hapten complex. After washing off the unbound reagent, the remaining tertiary complex is then exposed to light of the appropriate wavelength and the fluorescence observed indicates the presence of the antigen of interest.
  • Immunofluorescene and EIA techniques are both very well established in the art.
  • Other reporter molecules such as radioisotope, chemiluminescent or bioluminescent molecules, may also be employed.
  • the MUC genes of the present invention are likely to function in cell adhesion, signal transduction, growth regulation, epithelial cell protection and/or immunological reactions.
  • the classical gel-forming mucins function in protecting and lubricating epithelial tissues (particularly those of the respiratory and gastrointestinal tracts) by forming a layer of viscoelastic gel.
  • These new mucins, MUCH and MUC12 show structural similarity to MUC1.
  • MUC1 can be secreted, but unlike the classical mucins, it is primarily a type I transmembrane protein that interacts and complexes with other adhesion molecules, and is involved in signal transduction.
  • MUC12 has an EGF growth factor-like domain, is likely to be a transmembrane protein and has a putative tyrosine phosphorylation site that may participate in intracellular signalling. It is hypothesised that loss of MUC12 may be associated with poor prognosis in CRC.
  • the isolated MUC nucleic acids of the present invention are, therefore, considered in one embodiment, to correspond to cancer suppressor genes. Suppression may mean total inhibition of any development of large bowel cancer or a limitation of the severity of or an amelioration of the condition resulting from a large bowel cancer.
  • the MUC nucleic acids of the present invention are also considered in another embodiment to be capable of modulating disease conditions such as CRC, BC, IBD, CF, asthma, chronic bronchitis, ulcerative colitis and/or Crohn's disease
  • Cystic fibrosis (CF) is an inherited disease of epithelial cell chloride ion transport that affects multiple organ systems. It is the most common cause of severe, progressive lung disease and exocrine pancreatic insufficiency in childhood.
  • the cystic fibrosis transmembrane conductance regulator (CFTR) gene located on chromosome 7q22 encodes a large single chain protein that forms a chloride channel.
  • IBD Inflammatory bowel disease is characterised by considerable alterations in glycosylation, sialyation and sulphation of glycoproteins. It is unclear whether the changes in mucus production are a cause or response to the disease.
  • Susceptibility genes for inflammatory bowel disease have been localised to chromosomes 3, 12 and 7q22. Accordingly, the MUC genes of the present invention are considered candidates for susceptibility genes for IBD. Up or down regulation, or altered secretion of one of these mucins may influence the quality of colonic mucus and therefore the pathology of these diseases. Certain inherited forms of these genes may indicate a predisposition to IBD.
  • the identification of MUC genes and isolated MUC nucleic acids permits the generation of a range of therapeutic methods and compositions.
  • Such therapeutics may modulate MUC gene expression and the activity of MUC polypeptides.
  • Modulators contemplated by the present invention includes agonists and antagonists of MUC gene expression.
  • Antagonists of MUC gene expression include antisense molecules, ribozymes and co-suppression molecules.
  • Agonists include molecules which increase promoter activity or interfere with negative mechanisms.
  • Agonists of MUC include molecules which overcome any negative regulatory mechanism.
  • Antagonists of MUC polypeptides include antibodies and inhibitor peptide fragments.
  • Another class of therapeutics may be designed to mimic or block intracellular signal transduction by MUC polypeptides.
  • MUC functions as a suppressor of cancer development in the large bowel.
  • Hereditary cancers arise with loss of the wild-type gene.
  • germline mutations underlying large bowel cancer are inactivated for the MUC genes and, therefore, hereditary cancers have no functional copy of the gene.
  • sporadic large bowel cancers arise with somatic loss of both copies of the gene.
  • the present invention extends to the use of modulating levels of expression of MUC genes or their translation products in the context of cancers related thereto.
  • the present invention contemplates a method of gene therapy of a mammal.
  • a method of gene therapy of a mammal utilizes a gene therapy construct which includes an isolated MUC nucleic acid ligated into a gene therapy vector which provides one or more regulatory sequences that direct expression of said nucleic acid in said mammal.
  • Such regulatory sequences may include a promoter, an enhancer, a polyadenylation sequence, splice donor/acceptor sequences and translation termination and intiation sequences.
  • gene therapy vectors are derived from viral DNA sequences such as adenovirus, adeno-associated viruses, herpes-simplex viruses and retroviruses. Suitable gene therapy vectors currently available to the skilled person may be found in Robbins et al., 1998, Trends
  • Biotechnol. 16 35 for example, which is herein incorporated by reference.
  • one or more selected portions of a MUC nucleic acid may be oriented 3' ⁇ 5' in the gene therapy vector.
  • Administration of the gene therapy construct to said mammal may include delivery via direct oral intake, systemic injection, or delivery to selected tissue(s) or cells, or indirectly via delivery to cells isolated from the mammal or a compatible donor.
  • An example of the latter approach would be stem-cell therapy, wherein isolated stem cells having potential for growth and differentiation are transfected with the vector comprising the MUC nucleic acid. The stem-cells are cultured for a period and then transferred to the mammal being treated.
  • Delivery of said gene therapy construct to cells or tissues of said mammal or said compatible donor may be facilitated by microprojectile bombardment, liposome mediated transfection (e.g. lipofectin or lipofectamine), electroporation, calcium phosphate or DEAE-dextran- mediated transfection, for example.
  • liposome mediated transfection e.g. lipofectin or lipofectamine
  • electroporation calcium phosphate
  • DEAE-dextran- mediated transfection for example.
  • suitable delivery methods may be found in Chapter 9 of CURRENT PROTOCOLS IN MOLECULAR BIOLOGY (Eds. Ausubel et al.; John Wiley & Sons Inc., 1997 Edition), for example, which is herein incorporated by reference.
  • a MUC nucleic acid may be introduced into a cell to enhance the ability of that cell to survive, conversely, MUC antisense sequences such as 3' ⁇ 5' oligonucleotides may be introduced to decrease the survival capacity of any cell expressing an endogenous MUC gene.
  • MUC expression or activity is important in conditions of repressing cancer growth and/or development.
  • Decreased MUC expression or activity may be important, for example, in the treatment of cystic fibrosis or the treatment of inflammatory bowel disease.
  • the present invention contemplates a pharmaceutical composition comprising a MUC polypeptide or a derivative thereof or a modulator of MUC gene expression or activity, inclusive of anti- MUC antibodies.
  • active ingredients are referred to herein as the "active ingredients”, and are suitably provided in combination with one or more pharmaceutically-acceptable carriers and/or diluents.
  • the pharmaceutical forms suitable for injectable use include sterile aqueous solutions (where water soluble) and sterile powders for the extemporaneous preparation of sterile injectable solutions. It must be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms such as bacteria and fungi.
  • the carrier can be a solvent or, for example, water, ethanol, polyol (for example, glycerol, propylene glycol and liquid polyethylene glycol, and the like) or suitable mixtures thereof as well as vegetable oils.
  • the preventions of the action of microorganisms can be brought about by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, thirmersal and the like. In many cases, it will be preferable to include isotonic agents, for example, sugars or sodium chloride. Prolonged absorption of the injectable compositions can be brought about by the use in the compositions of agents delaying absorption, for example, aluminum monostearate and gelatin.
  • Sterile injectable solutions are prepared by incorporating the active compounds in the required amount in the appropriate solvent with various of the other ingredients enumerated above, as required, followed by filtered sterilization. In the case of sterile powders for the preparation of sterile injectable solutions, the preferred methods of preparation are vacuum drying and the freeze-drying technique which yield a powder of the active ingredient plus any additional desired ingredient from previously sterile-filtered solution thereof.
  • the active ingredients When the active ingredients are suitably protected they may be orally administered, for example, with an inert diluent or with an assimilable edible carrier, or it may be enclosed in hard or soft shell gelatin capsule, or it may be compressed into tablets, or it may be incorporated directly with the food of the diet.
  • the active compound For oral therapeutic administration, the active compound may be incorporated with excipients and used in the form of ingestible tablets, buccal tablets, troches, capsules, elixirs, suspensions, syrups, wafers, and the like.
  • Such compositions and preparations should contain at least 1 % by weight of active compound. The percentage of the compositions and preparations may, of course, be varied and may conveniently be between about 5 to about 80% of the weight of the unit. The amount of active compound in such therapeutically useful compositions in such that a suitable dosage will be obtained.
  • Preferred compositions or preparations according to the present invention are prepared so that an oral dosage unit form contains between about 0.1 ⁇ g and 2000
  • the tablets, troches, pills, capsules and the like may also contain the components as listed hereafter: a binder such as gum, acacia, corn starch or gelatin; excipients such as dicalcium phosphate; a disintegrating agent such as corn starch, potato starch, alginic acid and the like; a lubricant such as magnesium stearate; and a sweetening agent such a sucrose, lactose or saccharin may be added or a flavouring agent such as peppermint, oil of wintergreen, or cherry flavouring.
  • a binder such as gum, acacia, corn starch or gelatin
  • excipients such as dicalcium phosphate
  • a disintegrating agent such as corn starch, potato starch, alginic acid and the like
  • a lubricant such as magnesium stearate
  • a sweetening agent such as sucrose, lactose or saccharin may be added or a flavouring agent such as peppermint, oil of winter
  • tablets, pills, or capsules may be coated with shellac, sugar or both.
  • a syrup or elixir may contain the active compound, sucrose as a sweetening agent, methyl and propylparabens as preservatives, a dye and flavouring such as cherry or orange flavour.
  • any material used in preparing any dosage unit form should be pharmaceutically pure and substantially non-toxic in the amounts employed.
  • the active compound(s) may be incorporated into sustained-release preparations and formulations.
  • Pharmaceutically acceptable carriers and/or diluents include any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents and the like.
  • the use of such media and agents for pharmaceutically active substances is well known in the art. Except insofar as any conventional media or agent is incompatible with the active ingredient, use thereof in the therapeutic compositions is contemplated. Supplementary active ingredients can also be incorporated into the compositions.
  • Dosage unit form refers to physically discrete units suited as unitary dosages for the mammalian subjects to be treated; each unit containing a predetermined quantity of active material calculated to produce the desired therapeutic effect in association with the required pharmaceutical carrier.
  • the specification for the novel dosage unit forms of the invention are dictated by and directly dependent on (a) the unique characteristics of the active material and the particular therapeutic effect to be achieved, and (b) the limitations inherent in the art of compounding such an active material for the treatment of disease in living subjects having a diseased condition in which bodily health is impaired.
  • the principal active ingredient is compounded for convenient and effective administration in effective amounts with a suitable pharmaceutically acceptable carrier in dosage unit form as hereinbefore disclosed.
  • a unit dosage form can, for example, contain the principal active compound in amounts ranging from 0.5 ⁇ g to about 2000 mg. Expressed in proportions, the active compound is generally present in from about 0.5 ⁇ g to about 2000 mg/mL of carrier.
  • the dosages are determined by reference to the usual dose and manner of administration of the said ingredients. It is also convenient to represent the effective amounts of active ingredients as an amount per kg body weight. For example, the present invention encompasses effective amounts for 0.005 ⁇ g/kg body weight at 2000 mg/kg body weight.
  • the pharmaceutical composition may also comprise genetic molecules such as a vector capable of transfecting target cells where the vector carries a nucleic acid molecule capable of modulating MUC gene expression or MUC polypeptide activity.
  • the vector may, for example, be a viral vector.
  • the disease condition is selected from the group consisting of colorectal cancer (CRC), cystic fibrosis (CF), inflammatory bowel disease (IBD), breast cancer (BC), Crohn's disease, ulcerative colitis, asthma and chronic bronchitis.
  • CRC colorectal cancer
  • CF cystic fibrosis
  • IBD inflammatory bowel disease
  • BC breast cancer
  • Crohn's disease ulcerative colitis
  • asthma chronic bronchitis
  • the disease condition is selected from the group consisting of colorectal cancer (CRC), cystic fibrosis (CF), inflammatory bowel disease (IBD) and breast cancer (BC). although not limited thereto.
  • CRC colorectal cancer
  • CF cystic fibrosis
  • IBD inflammatory bowel disease
  • BC breast cancer
  • the therapeutic methods of the invention may therefore be used to alleviate one or more symptoms of diseases or be used as prophylactic treatments to prevent, or reduce the likelihood of, said symptoms from occurring.
  • the present invention is further described by the following non- limiting Examples.
  • Tissues were fixed for 24-48 hours in 10% v/v buffered formalin, dehydrated in ethanol, cleaned in chloroform and embedded in parraffin wax.
  • Biopsy specimens of normal colonic epithelium from four distinct regions of the colon were collected via colonoscopy from each of three healthy individuals undergoing routine colonoscopic screening.
  • intestinal biopsies were obtained via colonoscopy from ten patients with inflammatory bowel disease. Specimens were snap-frozen and stored at -70° C until RNA was extracted as per Example 3 below.
  • EXAMPLE 2 Cell Lines and Culture
  • LIM1215, LIM2405, LIM1863, LIM1899 (Ludwig Institute, Melbourne, Australia)
  • HT29 ATCC HTB38
  • SW480 ATCC CCL 2208
  • SW620 ATCC CCL 227).
  • LIM1215 and SW620 are each derived from CRC metastases.
  • Cell lines were maintained in RPM1 1640 with 10% v/v fetal calf serum, 2 mM glutamate, 25 mM HEPES, 60 mg/ml penicillin G and 100 mg/ml streptomycin sulfate and incubated in 5% v/v CO 2 and 95% v/v air at 37°C. Cultures were passaged twice weekly using standard techniques.
  • KPL-1 (a gift of Dr Junichi Kurebayashi, Suzuki, Japan)
  • MA11 (a gift of Dr Philip Rye, Oslo, Norway)
  • BT 20 DU4475
  • MCF-7 MDA-MB-453
  • SK-Br-3 T47D
  • UACC-893 ZR-75-1 and ZR-75-30
  • MDA-MB-435 and MDA-MB-468 (a gift of Dr. Janet Price, MD Anderson Cancer Center, Houston, TX).
  • RNA Extraction Total RNA was isolated by the method of Chomczynski and
  • RNA extraction buffer 4 M guanidinium isothiocyanate containing 25 mM sodium citrate, pH7.0, 0.5 % w/v sodium lauroyl sarcosine (SLS) and 0.1 M 2-mercaptoethanol. Tissue samples were homogenised in RNA extraction buffer. Extracted RNA was dissolved in RNase-free water and the concentration and purity determined by spectrophotometry at 260 and 280 nm (Sambrook et al., Molecular Cloning, A Laboratory Manual. 2nd Ed. Cold Spring Harbour Laboratory Press. Cold Spring Harbour, NY, 1989). The integrity of the RNA was assessed by denaturing agarose gel electrophoresis and samples transferred to HYBOND N (Amersham, Bucks, England) membrane by capillary blotting. EXAMPLE 4: DNA Sequencing
  • RNA sequencing reaction Approximately 500 ng of DNA were employed in a cycle sequencing reaction with 2.5 pmol of primer and 4 ⁇ l of Dye terminator or dRhodamine reaction mix (DNA Cycle Sequencing Kits, Perkin Elmer, Norwalk, CT,) in a total volume of 10 ⁇ l.
  • Reaction mixes contained Amplitaq DNA polymerase, dNTPs and fluorescently labelled dideoxynucleotides (dye terminators). Cycling reactions were as follows: 25 cycles of denaturation at 96°C (30 s), primer annealing at 50°C (15 s) and extension at 60°C (4 min). Unincorporated nucleotides were removed by ethanol precipitation.
  • One arbitrary 10mer primer (Operon Technologies Inc.) was selected at random to be employed in a PCR with the appropriate anchored primer.
  • Two patients, 101 and 112 were analyzed simultaneously and duplicates of two separate reverse transcription reactions electrophoresed on each gel. Gels were put down wet and autoradiographed for 1-3 days. DNA was removed from gel slices by boiling and reamplified by PCR. Bands were then cloned into pGEM-T (Promega Corporation, Madison, WI) and sequenced.
  • RNA from paired normal colonic mucosa and primary colorectal cancers were both amplified from normal colonic mucosal RNA of two patients and were consistently downregulated in the tumors from these patients in multiple PCR reactions (FIG. 1A).
  • discrete bands of approximately 720 bp for dd29 and 530 bp for dd34 were isolated and cloned into pGEM-T. Sequence analysis showed that both cDNAs were novel, with no match in any database accessed through the NCBI. Repetitive segments typical of mucin tandem repeats were observed in dd34.
  • EXAMPLE 6 Northern Blot Analysis
  • dd29 MUC12
  • dd34 MUCH
  • RNA samples were quantitated relative to a ⁇ 2 -microglobulin cDNA amplification control using densitometry. First strand cDNA synthesis was accomplished using 1 ⁇ g of total RNA.
  • PCR amplification of cDNA was performed in a total volume of 25 ⁇ l containing 1 ⁇ l of the first strand cDNA synthesis reaction products, 2.5 ⁇ l 10x Ta ⁇ f polymerase buffer (25 mM TAPS (tris-[hydroxymethyl]-methyl-amino- propane-sulfonic acid, sodium salt) pH 9.3, 50 mM KCI), 2 mM dNTPs, 25 mM MgCI 2 , 20 pmol each of the forward and reverse primers, and 2.5 U Ta ⁇ f polymerase.
  • Gene-specific forward and reverse primers for MUC12 and MUCH were designed to produce PCR products of 510 bp and 169 bp respectively.
  • Primers for ⁇ 2 -microglobulin generated a PCR product of 247 bp (Gussow ef a/., 1987, J. Immunol. 139 3132).
  • Primers were: MUC12F1 ; 5'-TGAAGGGCGACAATCTTCCTC-3' (SEQ ID NO:8); MUC12R1; 5'-TACACGAGGCTCTTGGCGATGTTG-3' (SEQ ID NO:9); MUC11 F1 ; 5'-CAGGCGTCAGTCAGGAATCTACAG-3' (SEQ ID NO: 10);
  • the amplification conditions were: 21 cycles of denaturation at 94°C (30 s) for MUC12, (24 cycles of denaturation at 94°C (30 s) for MUCH), annealing at 60°C (30 s) and extension at 72°C (30 s).
  • PCR products were electrophoresed on 1.2% 1x TBE gels and photographed. Due to the polydisperse signals obtained by Northern analysis, expression of MUCH and MUC12 was examined in a range of colorectal cancer cell lines and tissue mRNAs by multiplex semi-quantitative RT-PCR. dd29 was not expressed in any of six colorectal cancer cell lines examined (FIG. 1 D).
  • dd34 showed a different pattern of expression, with HT29, LIM1215, LIM1899, LIM1863 lines revealing very faint PCR products, and SW620 and SW480 lines showing relatively high levels of expression (FIG.1E).
  • downregulation was defined as amplified band intensity less than 30% of that observed from paired normal colon tissue.
  • dd29 was found to be downregulated or absent in 6/15 (40%) tumors with paired normal samples, and at low levels in 3/5 (60%) Dukes' stage D samples (where normal colon was not available for comparison) (FIG.1 F).
  • dd34 was downregulated in the tumors of 12/15 (80%) paired samples and expressed at low levels in 4/5 (80%) Dukes' stage D samples.
  • EXAMPLE 8 Differential Tissue Distribution of MUC11.
  • MUC12 and MUC3 mRNAs A human RNA "master blot" (Clontech, Palo Alto, CA, catalogue number 7770-1) with RNA from 50 different tissues and controls was used to examine mucin gene expression.
  • DNA fragments encoding dd29, dd34 and MUC3 (Genbank Accession No. M55405, a gift from Dr. Sandra Gendler, Mayo Clinic, Scotsdale, Arizona) were excised from vector and radiolabeled as described above. Hybridization was performed as per the manufacturer's instructions.
  • the master blot was reprobed with a radiolabeled ⁇ -actin cDNA as a loading control.
  • MUC11, MUC12 and MUC3 transcripts in RNA isolated from 50 different normal tissues showed a distinct pattern of expression for each gene (FIG. 5).
  • MUC12 and MUCH showed highest expression in colon but had different patterns in other organs, mainly restricted to those of epithelial type.
  • MUC11 had a wider epithelial distribution than MUC12 which was restricted to expression in the colon, and weakly in the pancreas, prostate and uterus.
  • MUC3 was found to be predominantly expressed in the small intestine and at much lower levels in the colon. Interestingly, it was also present in the thymus.
  • EXAMPLE 9 Extending the Seguences of dd29 and dd34
  • FIGS. 8A and 8B The strategy employed in the cloning of MUCH and MUC12 is shown in FIGS. 8A and 8B respectively.
  • a ⁇ gtl 1 human fetal brain 5'-STRETCH PLUS cDNA library (Clontech, Palo Alto, CA) was screened using radiolabeled dd29 and dd34. ⁇ DNA was extracted and inserts were excised, cloned into pBSK- and sequenced.
  • RT-PCR was performed on total RNA extracted from normal colon in a stringent touchdown PCR using high fidelity DyNAzyme DNA polymerase (Finnzymes, Espoo, Finland).
  • Primer combination MUC11 F1 and li5R (5'- GGGAACACTGTGGTTTCAGTTGAG-3'; SEQ ID NO: 16) yielded a PCR product of 2 kb demonstrating that these two cDNAs were derived from a single transcript. This product was cloned into pGEM-T and sequenced.
  • dd29F1 and dd29R1 9.3 PCR library screening - to extend sequence of dd29 Forward and reverse primers for dd29 (dd29F1 and dd29R1) were used in combination with a T7 vector-derived primer in a stringent touchdown PCR to screen an ulcerative colitis (UC) plasmid library (a gift from Dr. Jonathon Fawcett, Queensland Institute of Medical Research, Brisbane, Australia). Amplified products were purified, cloned into pGEM-T and sequenced.
  • UC ulcerative colitis
  • dd29 revealed that it was amplified as a result of priming of random 10mer at both ends of the PCR product and that it did not contain a 3' untranslated region (3'-UTR) or poly A tail.
  • primers MUC12TOTF1 and MUC12TOTR1 were designed to produce an expected PCR product of 1532 bp; primers corresponded to bases 230-250 and 1742-1762, respectively, in SEQ ID NO:6.
  • MUC12 contains two cysteine-rich EGF-like domains separated by a 150 amino acid non-mucin-like sequence (amino acids 261-410) containing five N-glycosylation sites and a potential coiled-coil domain.
  • the second cysteine-rich EGF-like domain is immediately followed by a putative transmembrane domain containing 26 hydrophobic or uncharged amino acids, and a cytoplasmic tail of 75 amino acids at the carboxyl terminus.
  • Muc3 (rMuc3), mouse Muc3 (mMuc3), human MUC4 (hMUC4) and rMuc4 is shown in FIG. 3.
  • MUC12 When aligned by the transmembrane amino acid sequences, MUC12 was found to have areas of significant homology to rMuc3, mMuc3 and hMUC3, including perfect conservation of eight cysteine residues in the second EGF-like domain. With inclusion of three small gaps, each of these cysteines also align with those in rat and human MUC4.
  • all six mucins contain a conserved EGF-like sequence of Cx(5)GPxCxCx(9)GExC.
  • Clone dd34 (544 bp) was also obtained as a result of priming of random 10mers at both ends of the PCR product.
  • Screening of a ⁇ gt11 human fetal brain library yielded two positive plaques which hybridized to dd34, clone Ii5 (1045 bp) and clone 2 (1043 bp). These two clones represented opposite ends of a 2.8 kb partial MUCH cDNA sequence (Genbank Accession Number AF147791 ), the linking of which was established by PCR (see Methods).
  • Conceptual translation of the MUC11 composite is shown in FIG. 4.
  • the entire 957 amino acid sequence consisted of serine, threonine and proline-rich tandem repeats of 28 amino acids in length, consistent with it being derived from a large epithelial mucin.
  • DNA fragments excised from dd29 (720 bp) and dd34 (530 bp) were nick translated with biotin-14-dATP and hybridized in situ at a final concentration of 10 ng/ ⁇ l to metaphases from two normal males.
  • the fluorescence in situ hybridization (FISH) method was modified from that previously described (Callen et al., 1990, Ann. Genet. 33 219) in that chromosomes were stained before analysis with both propidium iodide as counterstain and DAPI for chromosome identification. Images of metaphase preparations were captured by a cooled CCD camera using the CyroVision Ultra image collection and enhancement system (Applied Imaging Int Ltd, Sunderland, U.K.).
  • MUC11 CFHSRPASTHTTLFTED (SEQ ID NO: 17); corresponding to part of the degenerate tandem repeat region, specifically amino acid residues 690-705 deduced from the partial cDNA MUC11 clone, with an N-terminal cysteine residue added for conjugation); MUC12: TYRNFTEKMNDASSQEC (SEQ ID NO: 18); corresponding to part of the N- glycosylated region, specifically amino acid residues 286-302 deduced from the partial cDNA MUC12 clone, with a C-terminal cysteine residue added for conjugation).
  • KLH-conjugated peptide was diluted to 100 ⁇ g/mL in phosphate buffered saline (PBS) and mixed with an equal volume of complete Freund's adjuvant (CFA). Each mouse was injected intra-peritonealy with 0.5 mL of this mixture.
  • PBS phosphate buffered saline
  • CFA complete Freund's adjuvant
  • Each mouse was injected intra-peritonealy with 0.5 mL of this mixture.
  • Day 14 Each mouse was immunised as above but peptide was mixed with incomplete Freund's adjuvant (IFA).
  • IFA incomplete Freund's adjuvant
  • Day 33 Each mouse was immunised as on day 14.
  • Day 43 Each mouse was bled from the tail to assess antibody production by ELISA (see below).
  • Day 53 Each mouse was injected intra-venously with 100 ⁇ L of peptide at 100 ⁇ g/mL in PBS without adjuvant, and with 100 ⁇ L mixed with IFA intra-peritonealy.
  • Day 56 Mice were euthanased, and the spleen removed for fusion with myeloma cells.
  • Splenocytes were fused to Ag8 mouse myeloma cells at a ratio of 5:1 with polyethylene glycol using established methods (Harlow & Lane, supra).
  • Specific antibody producing clones were screened by a solid phase antigen antibody capture ELISA with the immunizing peptides bound to polystyrene assay plates using established methods (Harlow & Lane, supra). Positive clones were expanded, retested for specific antibody production and recloned by limiting dilution. Clones were further tested for reactivity with paraffin embedded normal colonic mucosa. 13.1 MUC 11 and MUC12 reactive hvbridomas Two hybridomas, one reacting with each of MUC11 and MUC12 peptides and with paraffin embedded colonic sections are described in Table 2.
  • Paraffin sections (4 ⁇ m) of normal colonic epithelium were dewaxed with xylene, hydrated in a graded series of ethanol to water and treated with 0.1 U/mL neuraminidase (Boehringer, Germany) in 50 mM Na acetate, 150 mM NaCl, 100 mM CaCI 2 buffer, pH 5.5 for 1 hr at room termperature to remove sialic acid groups. Sections were then treated with
  • M12.15 were semi-purified by PEG precipitation and diluted to 5-50 ⁇ g/mL in TBS/50% non-immune goat serum and incubated for 2 hours overnight at room temperature. Sections were washed once with 1 % TX-100 in TBS for
  • M11.9 reacts strongly with colonic epithelium, primarily with columnar cells of the surface epithelium (see FIG. 9A). Both goblet and columnar cells deep in the crypts are not stained by this antibody (see FIG. 9A). In surface epithelial columnar cells M11.9 reacted with the perinuclear cytoplasm, lateral cell membranes and most strongly as granular staining in the subapical cytoplasm (FIG. 9B).
  • M12.15 also reacts strongly with colonic epithelium, and like M11.9 it reacts primarily with columnar cells of the surface epithelium (see FIG. 9C). However, M12.15 gave a more diffuse cytoplasmic staining pattern than that seen with M11.9, although, like M11.9, the strongest staining was in the apical cytoplasm.
  • Immunohistochemistry in normal colonic mucosa with these antibodies demonstrates protein expression of the MUCH and MUC12 gene, supporting the mRNA studies.
  • the co-expression of MUC11 and MUC12 in normal colon is also consistent with the RT-PCR data showing similar levels of relative expression of these two mucin genes in different regions of the intestinal tract.
  • Riboprobes were made by in vitro transcription of DNA with SP6 and T7 RNA polymerases and incorporation of a digoxigenin-labelled uridine triphosphate (DIG-UTP). The orientation of inserts in pGEM-T was established by sequencing. Insert in the antisense direction and thus complementary to RNA template was the hybridisation probe and insert in the sense direction was used as a negative control.
  • DIG-UTP digoxigenin-labelled uridine triphosphate
  • 1 mg of purified linearised plasmid pGEM-T was labelled in the presence of 1/10 volume 10 x transcription buffer, 1/10 volume 10 x NTP mix (1 mM ATP, CTP, GTP, 0.65 mM UTP, 0.35 mM DIG-UTP), 10U RNase inhibitor and 40U of either SP6 or T7 RNA polymerase.
  • the reaction was carried out at 37°C for 2 hours and terminated by addition of 2 ⁇ l of 0.2M EDTA. Probes were ethanol precipitated with 1/11 volume 4M LiCI and placed at -20°C for 2 hours. They were then centrifuged at 12,000 g for 30 min at 4°C.
  • Sections were then incubated in proteinase K (5 mg/ml) at 37°C for 15 min and washed briefly in 3 x PBT. They were fixed in 4% paraformaldehyde for exactly 20 min and prehybridised for 4 hours at 70°C in hybridisation buffer (50% formamide, 5 x SSC, 1 % SDS, 500 mg/mL tRNA, 50 mg/mL heparin). Denatured probe (0.5 mg/section) was added to hybridation buffer and sections hybridised overnight at 70°C.
  • hybridisation buffer 50% formamide, 5 x SSC, 1 % SDS, 500 mg/mL tRNA, 50 mg/mL heparin.
  • Sections were washed in 2 x wash solution 1 (50% formamide, 5 x SSC, 1% SDS) at 65°C followed by 2 x washes in wash solution 2 (50% formamide, 2 x SSC) also at 65°C. Sections were then incubated with anti- digoxygenin-AP antibody at 1/2000 in PBS overnight at 40°C.
  • EXAMPLE 15 Expression of MUCH and MUCH in normal colon by RT-PCR The results of RT-PCR experiments to determine the expression patterns of MUCH and MUC12 genes in normal colonic epithelium are shown FIG. 10.
  • MUCH and MUC12 are predominantly expressed in the colon, although the data in FIG. 10 show that in fact their levels of expression vary within the colon. In this regard, a progressive increase (3-4 fold) in the expression of both MUCH and MUC12 was seen from the right colon to the rectum.
  • EXAMPLE 16 Expression of MUCH and MUC12 in CRC by RT-
  • MUC11 and MUC12 in CRC were investigated by RT-PCR, and the results are shown in FIG. 11. After 40 rounds of amplification, MUCH expression was observed in all CRC cell lines under investigation. Similarly, MUC12 expression was observed in all cell lines, although two cell lines, SW620 and SW116 revealed low levels of expression.
  • the results of Northern analysis with a dd34 (MUCH) probe showed that in nucleic acid extracts obtained from colonic tissue of four (4) of the (6) CRC patients tested, the level of MUC11 mRNA expression was lower relative to normal colonic tissue from the same patients. Similarly, MUC12 mRNA was downregulated in three (3) of five (5) CRC patients (data not shown).
  • Cytokeratin 20, (CK20) a colonic epithelial marker, was employed as a loading control due to the variable epithelial content of IBD tissues.
  • 'N' denotes tissues which appear macroscopically normal and 'D' refers to tissues reported to have IBD.
  • 'CA refers to the caecum, 'CO' the colon, 'LC the left colon, TC the transverse colon, 'RS' the recto-sigmoid colon, 'SI' the small intestine, 'IL'denotes the ileum and 'IP' an ileal pouch.
  • MUCH and MUC12 in BC tissue were investigated by RT-PCR, and the results are shown in FIG. 13. After 40 rounds of amplification, MUCH expression was identified in all breast cancer cell lines under investigation; at low levels in BT-20, DU4475, MDA-MB-435 and ZR-75-30 cell lines and at higher levels in the remaining nine cell lines.
  • MUC11 Eight of the cell lines showed MUC11 expression higher than the normal colonic cDNA positive control. MUCH is clearly highly expressed by most breast cancers and may impact upon the behaviour of the breast cancer cells. MUC11 may also be secreted by breast cancers and detection in serum could form the basis of diagnostic and prognostic testing for breast cancer.
  • mucin cDNAs mapped to 7q22 most likely represent separate genes, it is also possible that they are produced as a result of alternative mRNA splicing from a single, large mucin gene.
  • Northern blot analysis for MUCH, MUC12 and MUC3 shows that these encode large transcripts, estimated to be greater than 12 kb.
  • Multiple tissue RNA analysis showed no cross-reactivity between MUCH, MUC12 or MUC3.
  • MUCH and MUC12 showed predominant expression in the colon, while MUC3 was predominantly expressed in the small intestine and at very low levels in the colon. This expression pattern constitutes an important point of distinction between MUCH and MUC12 genes of the present invention and MUC3.
  • MUC11 and MUC12 are not homologous with any other human mucin genes, but show some degree of similarity within their variable tandem repeat regions to each other (71% over 653 bp). However, their clear differential expression patterns in normal and tumor tissues as well as tumor cell lines, show that they are distinct from each other, and from MUC3. While both MUC11 and MUC12 contain variable repeat regions typical of mucins, MUC12 is putatively a transmembrane mucin with features suggesting an involvement in growth regulation, a largely unrecognized function in human mucins. MUC12 is only the fourth human membrane- anchored epithelial mucin to be described to date, along with MUC1 , MUC3 and MUC4.
  • MUC1 has been shown to be involved in cell signaling via multiple tyrosine phosphorylation sites on its highly conserved cytoplasmic tail (Zrihan-Licht et al., 1994, FEBS Lett. 356 130). At its carboxyl terminus, MUC12 possesses a cytoplasmic tail containing a YNNF sequence (amino acids 557-560 in FIG. 2) which is similar to motifs recognized by SH2 domain-containing proteins (Songyang et al., Mol. Cell. Biol. 14 2777), suggesting that MUC12, like MUC1, may be involved in signal transduction.
  • the deduced amino acid sequence of the partial MUCH cDNA was composed entirely of serine/threonine-rich tandem repeats. There is a similarity between the tandem repeat consensus sequences of MUC11 (FIG. 4) and MUC12 (FIG. 2) and these also show limited homology to the MUC3 repeat (ITTETTSHSTPSFTSS). These similarities are consistent with evolution from a common ancestral gene. MUCH is more widely expressed than MUC12 and MUC3 however, with RNA detected in gastrointestinal, respiratory, reproductive and urinary tracts, and unexpectedly in the liver and thymus.
  • MUC11 and MUC12 are commonly downregulated in colorectal cancer suggesting they may play a role in epithelial cell growth modulation and/or differentiation.
  • downregulation appears to be so frequent, that it may be an early event in tumorigenesis.
  • MUCH and MUC12 genes on chromosome 7q22 it is possible that their expression is co-ordinately regulated and hence they are simultaneously downregulated in a large proportion of colorectal cancers.
  • Mucins are believed to protect epithelial cells from attack by pathogenic organisms and from mechanical and chemical damage. Therefore, reduced expression of these mucins could expose colonic epithelial cells to the harsh environment of the intestinal lumen. Furthermore, loss of a transmembrane mucin such as MUC12 may also contribute to loss of critical cell signaling.
  • chromosome 7q22 may have significance for two non-malignant epithelial diseases where aberrant mucin expression and/or function is a recognized component of pathology, namely, inflammatory bowel disease and cystic fibrosis.
  • Susceptibility genes for inflammatory bowel disease have been located to chromosomes 3, 12 and 7q22 (Satsangi et al., 1996, Nature Genet. 14 199).
  • MUCH and MUC12 must be considered candidates for involvement in inflammatory bowel disease given their chromosomal localization, expression in normal colon, and the documented alterations in mucins in this disease (Rhodes, 1997, QJM 90 79).
  • Mucins may also play a role in cystic fibrosis as patients with the same CFTR gene mutation do not demonstrate exactly the same phenotype in terms of mucus obstruction.
  • the existence of modifier genes has been postulated and mucin genes are obvious candidates (Harris & Reid, 1997, J. Med. Genet. 35 82).
  • a murine Mucin gene that shows C-terminal homology with MUC12 has recently been shown to be a major constituent of obstructive mucus in the gastrointestinal tract of mice with CF (Parmley et al., 1998, J. Clin. Invest 102 1798).
  • the CFTR gene lies in the adjacent chromosome band (7q31 ) to the MUC3, MUCH and MUC12 genes. While the significance of these findings is not clear, MUC11 and MUC12, which are expressed in many of the tissues affected by cystic fibrosis, should be considered as candidate modifier genes involved in the aetiology of this disease.
  • Mucins are encoded by large genes which have proved difficult to clone by conventional methods due to the repetitive nature of their tandem repeat regions.
  • the present inventors have unexpectedly identified by differential display two partial cDNAs which represent novel mucin genes that are predominantly expressed in colonic epithelium, both of which are downregulated in colorectal cancer.
  • MUCH and MUC 12 differ from the other mucin gene located on chromosome 7q22, MUC3.

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Abstract

L'invention concerne des acides nucléiques de MUC isolés correspondant à un gène Mucine situé sur le chromosome humain 7q22, ou sur un chromosome de mammalien structurellement ou fonctionnellement équivalent, ce gène Mucine étant normalement principalement exprimé dans le côlon. L'invention concerne également les utilisations diagnostiques et thérapeutiques d'acides nucléiques de MUC isolés, des polypeptides de MUC codés par ces acides nucléiques et des mAb anti-MUC.
PCT/AU1999/000579 1998-07-16 1999-07-16 Mucines WO2000004142A1 (fr)

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* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6818446B2 (en) 2001-11-21 2004-11-16 The Regents Of The University Of California Compositions and methods for the analysis of mucin gene expression and identification of drugs having the ability to inhibit mucin gene expression
WO2008147405A1 (fr) * 2006-04-05 2008-12-04 Oklahoma Medical Research Foundation Utilisation d'o-glycanes dans le traitement d'affections intestinales inflammatoires et de cancers

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
FOX M.F. ET AL: "Regional localizational of the intestinal mucin gene MUC3 to chromosome 7q22", ANN. HUM. GENET., vol. 56, 1992, pages 281 - 287 *
GUM J.R. ET AL: "Molecular Cloning of cDNAs Derived from a Novel Human Intestinal Mucin Gene", BIOCHEM. BIOPHYS. RES. COMMUN., vol. 171(1), 1990, pages 407 - 415 *

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6818446B2 (en) 2001-11-21 2004-11-16 The Regents Of The University Of California Compositions and methods for the analysis of mucin gene expression and identification of drugs having the ability to inhibit mucin gene expression
WO2008147405A1 (fr) * 2006-04-05 2008-12-04 Oklahoma Medical Research Foundation Utilisation d'o-glycanes dans le traitement d'affections intestinales inflammatoires et de cancers

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