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WO2003066869A1 - Gene suppresseur de tumeur lmt - Google Patents

Gene suppresseur de tumeur lmt Download PDF

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Publication number
WO2003066869A1
WO2003066869A1 PCT/AU2003/000126 AU0300126W WO03066869A1 WO 2003066869 A1 WO2003066869 A1 WO 2003066869A1 AU 0300126 W AU0300126 W AU 0300126W WO 03066869 A1 WO03066869 A1 WO 03066869A1
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Prior art keywords
seq
nucleotide sequence
sequence
conesponding
ribonucleotide
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PCT/AU2003/000126
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WO2003066869A8 (fr
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Wendy Dianne Cook
Benjamin John Mccaw
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The Walter And Eliza Hall Institute Of Medical Research
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Priority to AU2003202315A priority Critical patent/AU2003202315A1/en
Priority to US10/512,477 priority patent/US20050172424A1/en
Publication of WO2003066869A1 publication Critical patent/WO2003066869A1/fr
Publication of WO2003066869A8 publication Critical patent/WO2003066869A8/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
    • C07K14/4702—Regulators; Modulating activity
    • C07K14/4703—Inhibitors; Suppressors
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2217/00—Genetically modified animals
    • A01K2217/05—Animals comprising random inserted nucleic acids (transgenic)

Definitions

  • the present invention relates generally to the field of cancer therapy and cancer diagnostics and to agents useful therefor. More particularly, the present invention provides a novel tumor suppressor gene, an expression product including a polypeptide or protein encoded thereby and nucleic acid probes and primers and peptide fragments characteristic of the tumor suppressor gene or its expression product or mutants or derivatives thereof. The present invention further provides molecules generated through combinatorial chemistry or rational chemical synthesis or identified following screening of chemical libraries or natural sources which augment or otherwise facilitate the activity or function of the tumor suppressor gene or its expression product and in particular in cells which carry a deletion or other mutation including a gene silencing event in one or both alleles of the tumor suppressor gene.
  • the present invention provides diagnostic agents to detect the presence or absence of the tumor suppressor gene or the presence or absence of an expressible tumor suppressor gene or the presence or absence of an expression product of the tumor suppressor gene useful in determining the likelihood of development of a tumor in a vertebrate animal such as mammal and in particular a human.
  • the present invention further provides genetically modified animals carrying cells in which one or both alleles of the tumor suppressor gene are deleted or mutated or where expression of the gene is otherwise silenced.
  • tumors particularly important disease conditions include the development of tumors.
  • tumor as used in this context includes all forms of cancers such as carcinomas and sarcomas.
  • the development of tumors represents a major cause of mortality and morbidity in affected subjects not to mention the demands it causes on the health care system.
  • oncogenes oncogenesis
  • tumor suppressor genes A number of oncogenes and tumor suppressor genes have been characterized (Knudson, Nature Genet. 5: 103, 1993).
  • Such mutations and aberrations include both deletions and methylation of tumor suppressor genes.
  • Deletions may involve loss of a single allele, which is referred to as loss of heterozygosity (LOH) although homozygous deletion of both alleles may also occur. LOH is frequently associated with the loss of functional expression of the other allele.
  • LOH loss of heterozygosity
  • SEQ ID NO: Nucleotide and amino acid sequences are referred to by a sequence identifier number (SEQ ID NO:).
  • the SEQ ID NOs: correspond numerically to the sequence identifiers ⁇ 400>1 (SEQ ID NO:l), ⁇ 400>2 (SEQ ID NO:2), etc.
  • SEQ ID NO:1 sequence identifiers ⁇ 400>1
  • SEQ ID NO:2 sequence identifiers
  • a gene is identified having the characteristics of a human tumor suppressor gene with homologs in other animals such as mice.
  • the identification of this gene enables the development of diagnostic agents to detect aberrations in the gene or the ability of the gene to be expressed.
  • the gene may also be used to screen for genetic and non-genetic molecules which are capable of augmenting the tumor suppressor gene or its expression product in subjects where the gene is poorly expressed or where one or both alleles carry a mutation or have undergone an expression-silencing event.
  • the gene of the present invention is further useful in the identification of biochemical or genetic pathways comprising other potential targets which may influence cancer development. Microarray technology is particularly useful in this regard.
  • the present invention is predicated, therefore, in part on the identification of a tumor suppressor gene for a range of tumors including, most particularly, leukemia.
  • the tumor suppressor gene is referred to herein as "Lmt”.
  • the proteinaceous expression products of Lmt include a polypeptide referred to as "LMT”.
  • Nucleic acid expression products such as mRNA, RNA, introns and exons are encompassed by the term Lmt or is plural derivative Lmts.
  • the source of the Lmt gene is indicated by the first letter of the animal source such as "h t" for a human Lmt gene or "mLmt" for a murine Lmt gene.
  • a "polypeptide” includes a protein and a peptide.
  • the present invention further relates to the diagnosis and prognosis of cancer in a vertebrate animal by analysis of Lmt or LMT.
  • alterations in Lmt, expression of Lmt and/or the LMT expression product facilitates development of, or otherwise predisposes a subject to, the development of a tumor and in particular leukemia.
  • the term "tumor” includes all forms of cancer including sarcomas and carcinomas.
  • the present invention further relates to the treatment or prophylaxis of a tumor and in particular leukemia by the application of genetic therapy or the administration of proteinaceous or non-proteinaceous molecules which mimic or otherwise obviate the need for or otherwise act as agonists of Lmt or LMT.
  • Lmt or LMT or derivatives or homologs thereof may be employed to screen for potential therapeutic agents such as from natural sources or chemical or peptide libraries.
  • the present invention further provides an isolated nucleic acid molecule comprising all or part of Lmt.
  • Such nucleic acid molecules may be useful in genetic therapy to facilitate expression of genomic Lmt or to augment production of recombinant LMT.
  • the present invention further provides genetically modified cells including cells in which a recombinant Lmt gene or genetic agents to facilitate expression of genomic Lmt or to repair mutations in genomic Lmt have been introduced.
  • the present invention further provides methods of diagnosis of a tumor and in particular a leukemia by detecting aberrations in one or both alleles of the genomic Lmt gene, aberrations in the LMT or Lmt expression product or aberrations in the expression of one or both alleles of genomic Lmt.
  • the present invention provides, therefore, diagnostic agents such as primers or probes of the Lmt gene or mutated portions thereof and/or of the Lmt promoter region.
  • the diagnostic agents of the present invention extend to immunointeractive molecules and in particular antibodies to all or antigenic parts of LMT. Such diagnostic agents are useful in the detection of a tumor or a predisposition for development of a tumor and in particular leukemia.
  • the murine Lmt gene (mLmt) coding sequence is shown in SEQ ID NO:l.
  • the corresponding amino acid sequence for the mLMT polypeptide is shown in SEQ ID NO:2.
  • 5' and 3' untranslated regions of mLmt are represented in SEQ ID NO:3 and SEQ ID NO:4, respectively. Such 5' and 3' untranslated regions may be useful targets for diagnostic or genetic therapeutic purposes.
  • Introns may have a regulatory role in genetic regulation.
  • the nucleotide sequences for introns 1 to 8 of genomic mLmt are shown in SEQ ID NOs: 6 through 13, respectively.
  • the exons of mLmt are shown in SEQ ID NOs:14 through 22 corresponding to exons 1 to 10, respectively.
  • a nucleotide sequence encompassing or including all or part of the mLmt promoter is shown in SEQ ID NO: 3 and a reverse complement of the full length mLmt genomic sequence is represented in SEQ ID NO:5.
  • the human Lmt (hLmt) coding sequence is shown in SEQ ID NO:23 with the 5' untranslated region of the hLmt represented in SEQ ID NO:25 and the 3' untranslated region of hLmt represented in SEQ ID NO:26.
  • the reverse complement of genomic sequence of hLmt is shown in SEQ ID NO:27.
  • the amino acid sequence of hLMT is shown in SEQ ID NO:24.
  • introns 1 to 8 of hLmt are represented by SEQ ID NOs:28 through 35, respectively and exons 1 to 10 are represented in SEQ ID NOs:36 to 44, respectively.
  • Figure 1 is a diagrammatic representation of YACS and BACs showing particular markers identified over the deleted regions. Markers include MIT microsatellite markers, a polymorphism within the Midkine gene, unique BAC end sequences and novel polymorphisms. BACs are from the RPCI-23 library. Square brackets indicate the BACs encoding Lmt. Arrows indicate the positions of two novel polymorphisms, one of which (WC200/201) helped narrow the cdr.
  • Figure 2 is a photographic representation showing expression of mRNA for Lmt in normal tissues and non-deleted vs deleted tumour cell lines.
  • A RT-PCR of Lmt (upper panel) vs Madd (lower). Tracks 1,11, and 17: lkb size ladder, 2: brain, 3: C57BL/6 bone marrow, 4: BALB/c bone marrow, 5: heart, 6: kidney, 7: liver, 8: lung, 9: spleen, 10: thymus, 12: RSS1, 13: RSS4, 14: RSS5, 15: RSS7, 16: RSS8, 18: 13.1.1, 19: Stella, 20: .5bl2, 21: Rd5, 22: d2.2, 23: d2.2LRl.
  • C Northern blot of deleted ( ⁇ ) vs non- deleted cell lines.
  • Track 1 normal SJL/J brain, 2: bone marrow, 3: RSS1, 4: RSS4, 5: RSS5, 6: RSS7, 7: RSS8, 8: spleen, 9: 13.1.1, 10: Stella, 11: d2.2, 12: d2.2LRl, 13: RXS8, 14: RXS10, 15: RXS14, 16: RXS18, 17: RXS19, 18: RXS24, 19: RXS25, 20: RXS27, 21: RXS29, 22: RXS31, 23: RXS51.
  • RSS and RXS cell lines were established from murine primary myeloid leukemias generated in SJL/J and FI CBA x SJL/J mice, respectively.
  • Figure 3B is a representation showing a comparison of whole or partial coding regions of mammalian Lmt orthologs, and related sequences from C. elegans (unc86) and the primitive chordate C. intestinalis. For each sequence, accession numbers are given in brackets, followed by extents of nucleotide (N) and protein (P) homology.
  • Figure 4 is a photographic representation showing induction of Lmt expression via DNA demethylation with 5-Aza-2-deoxycytidine.
  • Cells were grown in 0.1 ⁇ M 5-Aza-2- deoxycytidine.
  • Expression of Lmt (upper panel) or control gene Gapdh (lower panel) was detected using Northern blot analysis of RNA made from treated and untreated cells.
  • Track 1 brain (positive control), 2: RXS51, 3,4: RSS1, 5,6: RSS5, 7,8: 13.1.1, 9,10: Stella, 11,12: .5bl2, 13,14: d2.2, 15,16: d2.2LRl.
  • Figure 5A is a photographic representation showing expression of Lmt in human 293T cells after transfection of an EGFP fusion construct.
  • RNA was extracted from cells transfected with either Lmt-EGFl? (left track) or the unmanipulated EGFP vector (right track), and the blot was probed with Lmt (upper panel) then with the control gene ⁇ -actin (lower panel). It can be seen that the Lmt mRNA is only detectable in the cells bearing the Lmt fusion construct.
  • Figure 5B is a graphical representation showing the relative rates of growth of the 293T cells transfected with E t-EGFP or EGFP alone.
  • the transfected cells were selected by sorting on a FACS II flow cytometer, and plated as described in Methods. On successive days cell densities were estimated by an MTS assay as described. As can be seen, whereas the cells expressing EGFP alone grew exponentially, expression of LMT-EGFP led to cessation of growth.
  • Figure 6 is a photographic representation showing a confocal image of a phalloidin stained 293-T cell expressing LMT-EGFP fusion protein (N-LMT-EGFP-C), 5 hours post transfection. As can be seen from the overlay image, the majority of the EGFP signal is excluded from the unstained nucleus.
  • Figure 7 is a similar photographic representation showing confocal images of a COS cell expressing LMT-EGFP fusion protein (N-LMT-EGFP), 24 hours post-transfection.
  • the image on the left is taken with Nomarski optics, to reveal cell morphology, the top right image reveals DAPI (4,6-diamidino-2-phenylindole) staining of DNA in the nucleus, and the bottom right image reveals EGFP -LMT, which can be seen in both the cytoplasm and the nucleus.
  • DAPI 4,6-diamidino-2-phenylindole
  • Figure 8 is a graphical representation of the decrease in expression of hLmt mRNA in CD 19+ blood cells of human patients suffering from Acute Myelogenous Leukemia (AML). Use was made of a commercially available array of cDNA from fractionated blood cells of normal individuals and patients with various blood diseases (Clontech Blood Disease Array).
  • each patient's cells were sorted according to cell surface markers.
  • cDNA was synthesized from each fraction, and dotted onto the array filter (6 dots per patient, each representing CD14+, CD19+, CD3+, mononuclear, polymorphic or unfractionated cells).
  • the array is divided into disease-specific groups (7-12 patients in each group).
  • the filter was probed with a h mt-specific sequence, followed after decay of the radioactivity by a ubiquitously expressed sequence (ubiquitin) as a loading control. After each probing, the filter was exposed to a phosphorimager, and the intensity of each spot on the resulting image was quantified using the Imagequant program. In order to normalize expression, the hLmt intensity for each spot was divided by its ubiquitin intensity. The only visible variation between groups of these normalized values was in the CD 19 fractions, where hLmt expression was suppressed in the AML patients, as shown in Figure 8A, which is a graphical representation of the data for CD 19 cells from all the patient groups, and Figure 8B, which shows data from AML and normal patients only. According to a 2-tailed student's t-test, the difference between the 2 groups is sigmficant, with a P value of 0.0001.
  • Figure 9 is a photographic representation of a polyacrylamide gel showing the separation of 293T cell proteins that had bound to purified recombinant LMT protein.
  • a fusion construct of murine LMT and glutathione transferase (GST) had been expressed in E. coli, and the protein had been purified from the lysed bacteria by affinity chromatography over a glutathione- sepharose column.
  • LMT protein was cleaved from the column and covalently attached to a second sepharose column by cyanogen bromide. Lysates from 293T cells were allowed to bind to (track 1) an uncoupled sepharose column or (track 3) a similar column to which LMT had been bound.
  • Track 2 is a second control track, for which the LMT-coupled column had been exposed to buffer instead of 293T cell lysate.
  • the two tracks at the left show protein molecular weight markers. It can be seen that track 3 contains proteins of several molecular weights that had bound specifically to the LMT protein, with proteins of 45 kilodaltons and 200 kilodaltons predominating.
  • the present invention provides an isolated nucleic acid molecule comprising a sequence of nucleotides which define a tumor suppressor gene (tsg).
  • the tsg of the present invention is referred to herein as "Lmt”.
  • Human Lmt is referred to as “hLmt” and murine Lmt is abbreviated to "mLmt”.
  • the proteinaceous expression product of Lmt is referred to herein as LMT (or hLMT or mLMT).
  • LMT or hLMT or mLMT
  • the abbreviation Lmts or LMTS is used to refer to more than one Lmt or LMT.
  • the expression product may also include RNA (including spliced introns and exons) and mRNA transcript and this is encompassed by the term "Lmt”.
  • a proteinaceous product includes a polypeptide comprising two or more amino acid residues.
  • a "polypeptide” in this context includes a protein or peptide. At least one allele of Lmt is deleted in some tumors with the second allele generally being silenced.
  • the Lmt gene is located on chromosome 2 in mice which corresponds to human chromosome llpll-12.
  • the present invention provides isolated nucleic acid molecules corresponding to Lmt as well as nucleic acid primers and probes useful for detecting Lmt, aberrations in the Lmt gene including deletions, insertions, additions and point mutations as well as gene silencing events.
  • the present invention further provides a means for detecting Lmt expression products including polypeptides and fragments thereof and in particular mutant or wild-type forms thereof.
  • the present invention further provides genetically modified animals having one or both alleles of Lmt or its homolog deleted, mutated or its/their expression silenced.
  • the present invention further provides genetically modified animals comprising Lmt genes introduced or deleted. Such animals are useful animal models for testing the pathogenesis of cancer and/or for testing potential anti-cancer agents.
  • one aspect of the present invention provides an isolated nucleic acid molecule comprising a sequence of nucleotides encoding or complementary to a sequence encoding the amino acid sequence set forth in SEQ ID NO:2 or SEQ ID NO:24 or an amino acid sequence having at least about 40% similarity to SEQ ID NO:2 or SEQ ID NO:24 after optimal alignment.
  • SEQ ID NO:2 represents a polypeptide expression product of mLmt and is included in the term mLMT.
  • SEQ ID NO:24 represents a polypeptide expression product of hLmt and is included in the term hLMT.
  • LMT is proposed, in accordance with the present invention, to be a tumor suppressor polypeptide and facilitates inhibition or suppression of tumor development.
  • the isolated nucleic acid molecule, therefore, and nucleic acid expression products are refened to as Lmt.
  • an isolated nucleic acid molecule comprising a sequence of nucleotides encoding or complementary to a sequence encoding a polypeptide capable of inhibiting, preventing or otherwise suppressing development of a tumor in a vertebrate animal, said polypeptide having the identifying characteristics of LMT including an amino acid sequence as set forth in SEQ ID NO:2 or SEQ ID NO:24 or an amino acid sequence having at least about 40% similarity to SEQ ID NO:2 or SEQ ID NO:24 after optimal alignment.
  • the isolated nucleic acid molecule is referred to as Lmt.
  • an even more particular aspect of the present invention contemplates an isolated nucleic acid molecule comprising a nucleotide sequence or a complementary nucleotide sequence having the identifying characteristics of Lmt including a nucleotide sequence substantially as set forth in SEQ ID NO:l or SEQ ID NO:23 or a nucleotide sequence having at least about 40% similarity to SEQ ID NO:l or SEQ ID NO:23 after optimal alignment or a nucleotide sequence capable of hybridizing to SEQ ID NO:l or SEQ ID NO:23 or its complementary form under low stringency conditions.
  • Lmt includes a cDNA or mRNA sequence comprising an open reading frame or a genomic nucleotide sequence comprising exons and optionally introns. Consequently, another aspect of the present invention provides an isolated nucleic acid molecule comprising a sequence of nucleotides selected from the list comprising:-
  • xlii a nucleotide sequence of reverse complement genomic hLmt (SEQ ID NO:27);
  • xliii a nucleotide sequence capable of hybridizing to any one of (i) to (xlii) or their complementary forms thereof under low stringency conditions;
  • (xliv) a nucleotide sequence having at least about 40% similarity to any one of (i) to (xlii) after optimal alignment.
  • Lmt includes derivatives or homologs thereof.
  • a derivative includes a mutant, part, fragment or portion of Lmt as well as any fusion derivatives such as formed by the fusion of one or more nucleotide sequences to the 5' or 3' terminal portion of Lmt or to the 5' or 3' tenninal portion of a fragment of Lmt such as an intron or exon fragment.
  • a derivative of Lmt includes single or multiple nucleotide substitutions, additions and or deletions or inversions to all or part of Lmt and also includes point mutations and cross-over events.
  • Lmt also includes its 5' and 3' untranslated regions including a promoter operably linked to Lmt.
  • the mLmt promoter is defined by SEQ ID NO:3.
  • the hLmt promoter is defined by SEQ ID NO:25.
  • nucleic acid molecule comprising a promoter which nucleic acid molecule comprises the nucleotide sequence substantially as set forth in SEQ ID NO:3 or SEQ ID NO:25 or a nucleotide sequence having at least about 40% similarity thereto or a nucleotide sequence capable of hybridizing to SEQ JD NO:3 or SEQ ID NO:25 or its complementary sequence under low stringency conditions.
  • the Lmt gene may be from an animal cell including from a mammal, insect, reptile, fish (including D. rerio, zebrafish), avian species, arachnid or lower order organism such as a yeast, fungus or C. elegans.
  • the Lmt gene is from a mammal such as a human, primate, livestock animal (e.g. sheep, pig, cow, donkey, horse, goat), laboratory test animal (e.g. mouse, rat, rabbit, guinea pig), a companion animal (e.g. dog, cat) or a captured wild animal (e.g. rodents, foxes, deer, kangaroo).
  • the Lmt gene is from a human or mouse.
  • Lmt nucleotide sequences are of human and murine origin. However, the present invention extends to other sources of Lmt such as from zebrafish. Reference herein to "Lmt” includes reference to the human or murine forms of the gene as well as homologs from other species such as C. elegans and zebrafish.
  • gene is used in its broadest sense and includes cDNA corresponding to the exons of a gene. Accordingly, reference herein to a “gene” is to be taken to include:-
  • a classical genomic gene consisting of transcriptional and/or translational regulatory sequences and or a coding region and/or non-translated sequences (i.e. introns, 5'- and
  • gene is also used to describe synthetic or fusion molecules encoding all or part of an expression product.
  • nucleic acid molecule and “gene” may be used interchangeably.
  • polynucleotide may also be used to describe a nucleic acid molecule, nucleotide sequence and a gene.
  • similarity includes exact identity between compared sequences at the nucleotide or amino acid level. Where there is non-identity at the nucleotide level, "similarity” includes differences between sequences which result in different amino acids that are nevertheless related to each other at the structural, functional, biochemical and/or conformational levels. Where there is non-identity at the amino acid level, “similarity” includes amino acids that are nevertheless related to each other at the structural, functional, biochemical and/or conformational levels. In a particularly preferred embodiment, nucleotide and sequence comparisons are made at the level of identity rather than similarity.
  • references to describe sequence relationships between two or more polynucleotides or polypeptides include “reference sequence”, “comparison window”, “sequence similarity”, “sequence identity”, “percentage of sequence similarity”, “percentage of sequence identity”, “substantially similar” and “substantial identity”.
  • a “reference sequence” is at least 12 but frequently 15 to 18 and often at least 25 or above, such as 30 monomer units, inclusive of nucleotides and amino acid residues, in length. Because two polynucleotides may each comprise (1) a sequence (i.e.
  • sequence comparisons between two (or more) polynucleotides are typically perfonned by comparing sequences of the two polynucleotides over a "comparison window" to identify and compare local regions of sequence similarity.
  • a “comparison window” refers to a conceptual segment of typically 12 contiguous residues that is compared to a reference sequence.
  • the comparison window may comprise additions or deletions (i.e. gaps) of about 20% or less as compared to the reference sequence (which does not comprise additions or deletions) for optimal alignment of the two sequences.
  • Optimal alignment of sequences for aligning a comparison window may be conducted by computerised implementations of algorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software Package Release 7.0, Genetics Computer Group, 575 Science Drive Madison, WI, USA) or by inspection and the best alignment (i.e. resulting in the highest percentage homology over the comparison window) generated by any of the various methods selected.
  • GAP Garnier et al.
  • Altschul et al. Nucl Acids Res. 25: 3389, 1997.
  • a detailed discussion of sequence analysis can be found in Unit 19.3 of Ausubel et al. ("Current Protocols in Molecular Biology" John Wiley & Sons Inc, 1994-1998, Chapter 15).
  • sequence similarity and “sequence identity” as used herein refer to the extent that sequences are identical or functionally or structurally similar on a nucleotide-by-nucleotide basis or an amino acid-by-amino acid basis over a window of comparison.
  • a “percentage of sequence identity” is calculated by comparing two optimally aligned sequences over the window of comparison, determining the number of positions at which the identical nucleic acid base (e.g. A, T, C, G, 1) or the identical amino acid residue (e.g.
  • sequence identity will be understood to mean the "match percentage” calculated by the D ⁇ ASIS computer program (Version 2.5 for windows; available from Hitachi Software engineering Co., Ltd., South San Francisco, California, USA) using standard defaults as used in the reference manual accompanying the software. Similar comments apply in relation to sequence similarity.
  • the percentage similarity between a particular sequence and a reference sequence is at least about 50% or at least about 60% or at least about 70% or at least about 80% or at least about 90% or at least about 95% or above such as at least about 96%, 97%, 98%, 99% or greater.
  • Reference herein to a low stringency includes and encompasses from at least about 0 to at least about 15% v/v formamide and from at least about 1 M to at least about 2 M salt for hybridization, and at least about 1 M to at least about 2 M salt for washing conditions.
  • low stringency is at from about 25-30°C to about 42°C. The temperature may be altered and higher temperatures used to replace formamide and/or to give alternative stringency conditions.
  • Alternative stringency conditions may be applied where necessary, such as medium stringency, which includes and encompasses 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 hybridization, and at least about 0.5 M to at least about 0.9 M salt for washing conditions, or high stringency, which 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 hybridization, and at least about 0.01 M to at least about 0.15 M salt for washing conditions.
  • medium stringency which includes and encompasses 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 hybridization, and at least about 0.5 M to at least about 0.9 M salt for washing conditions
  • high stringency which includes and encompasses from at least about 31% v/v to at least about 50% v/v form
  • T m of a duplex DNA decreases by 1°C with every increase of 1% in the number of mismatch base pairs (Bonner and Laskey, Eur. J. Biochem. 46: 83, 1974).
  • Formamide is optional in these hybridization conditions. Accordingly, particularly preferred levels of stringency are defined as follows: low stringency is 6 x SSC buffer, 0.1 % w/v SDS at 25-42°C; a moderate stringency is 2 x SSC buffer, 0.1% w/v SDS at a temperature in the range 20°C to 65°C; high stringency is 0.1 x SSC buffer, 0.1% w/v SDS at a temperature of at least 65°C.
  • Lmt allele refers to normal alleles of the Lmt locus as well as alleles carrying variations that predispose individuals to development of a tumor.
  • nucleic acids include RNA, cDNA, genomic DNA, synthetic forms and mixed polymers, both sense and antisense strands, and may be chemically or biochemically modified or may contain non-natural or derivatized nucleotide bases, as will be readily appreciated by those skilled in the art.
  • modifications include, for example, labels, methylation, substitution of one or more of the naturally occurring nucleotides with an analog (such as the morpholine ring), internucleotide modifications such as uncharged linkages (e.g. methyl phosphonates, phosphotriesters, phosphoamidates, carbamates, etc.), charged linkages (e.g.
  • phosphorothioates phosphorodithioates, etc.
  • pendent moieties e.g. polypeptides
  • intercalators e.g. acridine, psoralen, etc.
  • chelators e.g. acridine, psoralen, etc.
  • alkylators e.g. ⁇ -anomeric nucleic acids, etc.
  • synthetic molecules that mimic polynucleotides in their ability to bind to a designated sequence via hydrogen binding and other chemical interactions. Such molecules are known in the art and include, for example, those in which peptide linkages substitute for phosphate linkages in the backbone of the molecule.
  • the present invention further provides recombinant nucleic acids including a recombinant construct comprising all or part of the Lmt region.
  • the recombinant construct may be capable of replicating autonomously in a host cell. Alternatively, the recombinant construct may become integrated into the chromosonal DNA of the host cell.
  • Such a recombinant polynucleotide comprises a polynucleotide of genomic, cDNA, semi-synthetic or synthetic origin which, by virtue of its origin or manipulation: (i) is not associated with all or a portion of a polynucleotide with which it is associated in nature; (ii) is linked to a polynucleotide other than that to which it is linked in nature; or (iii) does not occur in nature.
  • nucleic acids according to the invention include RNA, reference to the sequence shown should be construed as reference to the RNA equivalent with U substituted for T.
  • nucleic acids comprising sequences otherwise not naturally occurring are provided by the present invention.
  • wild-type sequence may be employed, it will often be altered, e.g. by deletion, substitution or insertion.
  • cDNA or genomic libraries of various types may be screened as natural sources of the nucleic acids of the present invention or such nucleic acids may be provided by amplification of sequences resident in genomic DNA or other natural sources, e.g. by PCR.
  • the choice of cDNA libraries normally corresponds to a tissue source which is abundant in mRNA for the desired protein.
  • Phage libraries are normally prefened but other types of libraries may be used. Clones of a library are spread onto plates, transferred to a substrate for screening, denatured and probed for the presence of desired sequences.
  • Lmt locus and Lmt allele refer to the double-stranded DNA comprising the locus, allele or region, as well as either of the single-stranded DNAs comprising the locus, allele or region.
  • a "portion or part or fragment" of the Lmt locus or allele is defined as having a minimal size of at least about 10 nucleotides or preferably about 13 nucleotides or more preferably at least about 20 nucleotides and may have a minimal size of at least about 35 nucleotides. This definition includes all sizes in the range of 10-35 nucleotides as well as greater than 35 nucleotides.
  • this definition includes nucleic acids of 12, 15, 20, 25, 40, 60, 80, 100, 200, 300, 400 or 500 nucleotides or nucleic acids having any number of nucleotides within these values (e.g. 13, 16, 23, 30, 28, 50, 72, 121, etc. nucleotides) or nucleic acids having more than 500 nucleotides or any number of nucleotides between 500 and the number shown in SEQ ID NO:l or SEQ ID NO:5 or SEQ ID NO:23 or SEQ ID NO:27.
  • the present invention includes all novel nucleic acids having at least 10 nucleotides derived from SEQ ID NO:l, SEQ ID NO:5, SEQ ID NO:23 or SEQ ID NO:27 or a complement or functional equivalent thereof.
  • the present invention does not encompass nucleic acids of the prior art although known nucleic acids useful in detecting or treating Lmt- suppressed tumors are contemplated by the present invention.
  • “Operably linked” refers to a juxtaposition wherein the components so described are in a relationship permitting them to function in their intended manner.
  • a promoter is operably linked to a coding sequence if the promoter effects its transcription or expression.
  • polynucleotides of the present invention may be produced by replication in a suitable host cell. Natural or synthetic polynucleotide fragments coding for a desired fragment will be incorporated into recombinant polynucleotide constructs, usually DNA constructs, capable of introduction into and replication in a prokaryotic or eukaryotic cell. Usually the polynucleotide constructions will be suitable for replication in a unicellular host, such as yeast or bacteria, but may also be intended for introduction to (with or without integration within the genome) cultured mammalian or plant or other eukaryotic cell lines. The purification of nucleic acids produced by the methods of the present invention are described, e.g. in Sambrook et al.
  • the polynucleotides of the present invention may also be produced by chemical synthesis, e.g. by the phosphoramidite method described by Beaucage and Carruthers (Tetra. Letts. 22: 1859- 1862, 1981) or the triester method according to Matteucci et al. (J Am. Chem. Soc. 103: 3185, 1981) and may be performed on commercial, automated oligonucleotide synthesizers.
  • a double-stranded fragment may be obtained from the single-stranded product of chemical synthesis either by synthesizing the complementary strand and annealing the strands together under appropriate conditions or by adding the complementary strand using DNA polymerase with an appropriate primer sequence.
  • Polynucleotide constructs prepared for introduction into a prokaryotic or eukaryotic host may comprise a replication system recognized by the host, including the intended polynucleotide fragment encoding the desired polypeptide and will preferably also include transcription and translational initiation regulatory sequences operably linked to the polypeptide encoding segment.
  • Expression vectors may include, for example, an origin of replication or autonomously replicating sequence (ARS) and expression control sequences, a promoter, an enhancer and necessary processing information sites, such as ribosome-binding sites, RNA splice sites, polyadenylation sites, transcriptional terminator sequences and mRNA stabilizing sequences.
  • ARS origin of replication or autonomously replicating sequence
  • Secretion signals may also be included where appropriate which allow the protein to cross and/or lodge in cell membranes or be secreted from the cell.
  • Such vectors may be prepared by means of standard recombinant techniques well known in the art and discussed, for example, in Sambrook et al (1989; supra) or Ausubel et al (1994-98; supra).
  • an appropriate promoter and other necessary vector sequences will be selected so as to be functional in the host and may include, when appropriate, those naturally associated with Lmt genes. Examples of workable combinations of cell lines and expression vectors are described in Sambrook et al (1989) or Ausubel et al. (1994-98, supra). Many useful vectors are known in the art and may be obtained from such vectors as Stratagene, New England Biolabs, Promega Biotech and others. Promoters such as the trp, lac and phage promoters, tRNA promoters and glycolytic enzyme promoters may be used in prokaryotic hosts.
  • Useful yeast promoters include promoter regions for metallothionein, 3-phosphoglycerate kinase or other glycolytic enzymes such as enolase or glyceraldehyde-3-phosphate dehydrogenase, enzymes responsible for maltose and galactose utilization and others. Vectors and promoters suitable for use in yeast expression are further described in European Patent Publication No. 0 073 675.
  • Non-native mammalian promoters might include the early and late promoters from SN40 (Fiers et al, Nature 273: 113-120, 1978) or promoters derived from murine molony leukemia virus, mouse tumor virus, avian sarcoma viruses, adenovirus II, bovine papilloma virus or polyoma. Insect promoters may be derived from baculovirus.
  • the construct may be joined to an amplifiable gene (e.g. DHFR) so that multiple copies of the gene may be made.
  • an amplifiable gene e.g. DHFR
  • Enhancers and Eukaryotic Gene Expression Cold Spring Harbor Press, Cold Spring Harbour, New York (1983). See also, e.g. U.S. Patent No. 5,691,198.
  • expression vectors may replicate autonomously, they may also replicate by being inserted into the genome of the host cell, by methods well known in the art.
  • Expression and cloning vectors will likely contain a selectable marker and a gene encoding a protein necessary for survival or growth of a host cell transformed with the vector. The presence of this gene ensures growth of those host cells which express the inserts.
  • Typical selection genes encode proteins that (a) confer resistance to antibiotics or other toxic substances, e.g. ampicillin, neomycin, methotrexate, etc., (b) complement auxotrophic deficiencies, or (c) supply critical nutrients not available from complex media, e.g. the gene encoding D-alanine racemase for Bacillus.
  • the choice of the proper selectable marker will depend on the host cell and appropriate markers for different hosts are well known in the art.
  • the vectors containing the nucleic acids of interest can be transcribed in vitro and the resulting RNA introduced into the host cell by well-known methods, e.g. by injection (see Kubo et al, FEBS Lett. 241: 119, 1988), or the vectors can be introduced directly into host cells by methods well known in the art, which vary depending on the type of cellular host, including electroporation; transfection employing calcium chloride, rubidium chloride, calcium phosphate, DEAF-dextran, or other substances; microprojectile bombardment; lipofection; infection (where the vector is an infectious agent, such as a retroviral genome); and other methods. See generally, Sambrook et al. (1989; supra) and Ausubel et al.
  • nucleic acids and polypeptides (see below) of the present invention may be prepared by expressing the Lmt nucleic acids or portions thereof in vectors or other expression vehicles in compatible prokaryotic or eukaryotic host cells.
  • prokaryotic hosts are strains of E. coli, although other prokaryotes, such as Bacillus subtilis or Pseudomonas may also be used.
  • Mammalian or other eukaryotic host cells such as those of yeast, filamentous fungi, plant, insect or amphibian or avian species, may also be useful for production of the proteins of the present invention.
  • Propagation of rnqmmalian cells in culture is per se well known. See, Jakoby and Pastan (eds.), Cell Culture. Methods in Enzymology, Nol. 58, 1979 (Academic Press, Inc., Harcour Brace Jovanovich (New York).
  • Examples of commonly used mammalian host cell lines are NERO and HeLa cells, Chinese hamster ovary (CHO) cells, and WI38, BHK and COS cell lines.
  • An example of a commonly used insect cell line is SF9.
  • other cell lines may be appropriate, e.g. to provide higher expression, desirable glycosylation patterns or other features.
  • Clones are selected by using markers depending on the mode of the vector construction.
  • the marker may be on the same or a different D ⁇ A molecule, preferably the same D ⁇ A molecule.
  • the transformant may be selected, e.g. by resistance to ampicillin, tetracycline or other antibiotics. Production of a particular product based on temperature sensitivity may also serve as an appropriate marker.
  • Prokaryotic or eukaryotic cells transformed with the polynucleotides of the present invention will be useful not only for the production of the nucleic acids and polypeptides of the present invention but also, for example, in studying the characteristics of a LMT polypeptide.
  • Antisense polynucleotide sequences are useful in preventing or diminishing the expression of the Lmt locus, as will be appreciated by those skilled in the art, although this is unlikely to be necessary unless Lmt is over-expressed and is causing collateral, physiological or genetic problems.
  • Polynucleotide vectors for example, containing all or a portion of the Lmt locus or other sequences from the Lmt region (particularly those flanking the Lmt locus) may be placed under the control of a promoter in an antisense orientation and introduced into a cell. Expression of such an antisense construct within a cell will interfere with Lmt transcription and/or translation. Furthermore, co-suppression and mechanisms to induce RNAi may also be employed. Such techniques may be useful to inhibit genes which negatively control Lmt expression.
  • morpholinos are oligonucleotides composed of morpholine nucleotide derivatives and phosphorodiamidate linkages (for example, Summerton and Weller, Antisense and Nucleic Acid Drug Development 7: 187-195, 1997).
  • Such compounds are injected into embryos such as zebrafish embryos, where they are taken up efficiently by cells, and the effect of interference with mRNA is seen within hours. It is predicted that such experiments using antisense Lmt morpholinos would result in disruption of normal hemopoiesis, and possibly excessive growth of myeloid cells.
  • the present invention is particularly useful for screening compounds by using the LMT polypeptide or binding fragment thereof in any of a variety of drug screening techniques, such as those described herein and in International Publication No. WO 97/02048.
  • an expression product of Lmt may be a polypeptide encoded by the open reading frame of Lmt (i.e. see SEQ ID NO:2 or SEQ ID NO:24) or it may be an mRNA or RNA molecule spliced from an mRNA or precursor form thereof.
  • RNA molecules include exons and introns.
  • Such molecules or their conesponding DNA sequences may be useful as regulatory molecules in their own right or as diagnostic probes or primers for detecting wild-type or a mutated or deleted Lmt gene.
  • an expression product from Lmt said expression product selected from the list comprising:-
  • a ribonucleotide sequence conesponding to SEQ ID NO:3 (vi) a ribonucleotide sequence conesponding to SEQ ID NO:3; (vii) a ribonucleotide sequence conesponding to SEQ ID NO:4 or a nucleotide sequence having at least about 40% similarity to SEQ ID NO:4 after optimal alignment or a nucleotide sequence capable of hybridizing to SEQ ID NO:4 or its complementary form under low stringency conditions;
  • xi a ribonucleotide sequence conesponding to SEQ ID NO:6 or a nucleotide sequence having at least about 40% similarity to SEQ ID NO: 6 after optimal alignment or a nucleotide sequence capable of hybridizing to SEQ ID NO:6 or its complementary form under low stringency conditions;
  • xiii a ribonucleotide sequence conesponding to SEQ ID NO: 7 or a nucleotide sequence having at least about 40% similarity to SEQ ID NO: 7 after optimal alignment or a nucleotide sequence capable of hybridizing to SEQ ID NO: 7 or its complementary form under low stringency conditions;
  • xv a ribonucleotide sequence corresponding to SEQ ID NO: 8 or a nucleotide sequence having at least about 40% similarity to SEQ ID NO: 8 after optimal alignment or a nucleotide sequence capable of hybridizing to SEQ ID NO:8 or its complementary form under low stringency conditions;
  • (xix) a ribonucleotide sequence conesponding to SEQ JD NO: 10 or a nucleotide sequence having at least about 40% similarity to SEQ ID NO: 10 after optimal alignment or a nucleotide sequence capable of hybridizing to SEQ JD NO: 10 or its complementary form under low stringency conditions;
  • xli a ribonucleotide sequence conesponding to SEQ ID NO:21 or a nucleotide sequence having at least about 40% similarity to SEQ ID NO:21 after optimal alignment or a nucleotide sequence capable of hybridizing to SEQ ID NO:21 or its complementary form under low stringency conditions;
  • (xliii) a ribonucleotide sequence conesponding to SEQ ID NO: 22 or a nucleotide sequence having at least about 40% similarity to SEQ ED NO:22 after optimal alignment or a nucleotide sequence capable of hybridizing to SEQ ID NO:22 or its complementary form under low stringency conditions;
  • xlv a ribonucleotide sequence conesponding to SEQ ED NO:23 or a nucleotide sequence having at least about 40% similarity to SEQ ED NO:23 after optimal alignment or a nucleotide sequence capable of hybridizing to SEQ ED NO:23 or its complementary form under low stringency conditions;
  • xlvii a ribonucleotide sequence conesponding to SEQ ED NO:25 or a nucleotide sequence having at least about 40% similarity to SEQ ED NO:25 after optimal alignment or a nucleotide sequence capable of hybridizing to SEQ ED NO:25 or its complementary form under low stringency conditions;
  • xlviii a ribonucleotide sequence conesponding to SEQ ID NO:25;
  • xlix a ribonucleotide sequence conesponding to SEQ ED NO:26 or a nucleotide sequence having at least about 40% similarity to SEQ JD NO:26 after optimal alignment or a nucleotide sequence capable of hybridizing to SEQ ED NO:26 or its complementary form under low stringency conditions;
  • a ribonucleotide sequence conesponding to SEQ ED NO:29 (lvi) a ribonucleotide sequence conesponding to SEQ ED NO:29; (lvii) a ribonucleotide sequence conesponding to SEQ ED NO:30 or a nucleotide sequence having at least about 40% similarity to SEQ ED NO:30 after optimal alignment or a nucleotide sequence capable of hybridizing to SEQ ED NO:30 or its complementary form under low stringency conditions;
  • lix a ribonucleotide sequence conesponding to SEQ ED NO:31 or a nucleotide sequence having at least about 40% similarity to SEQ ED NO:31 after optimal alignment or a nucleotide sequence capable of hybridizing to SEQ ED NO:31 or its complementary form under low stringency conditions;
  • (lxiii) a ribonucleotide sequence conesponding to SEQ ED NO:33 or a nucleotide sequence having at least about 40% similarity to SEQ ED NO:33 after optimal alignment or a nucleotide sequence capable of hybridizing to SEQ ED NO:33 or its complementary form under low stringency conditions;
  • (lxix) a ribonucleotide sequence conesponding to SEQ ED NO: 36 or a nucleotide sequence having at least about 40% similarity to SEQ ED NO:36 after optimal alignment or a nucleotide sequence capable of hybridizing to SEQ ED NO:36 or its complementary form under low stringency conditions;
  • LMT protein or “LMT polypeptide” or specific LMT molecules from human (hLMT) or murine (mLMT) sources refers to a protein including a polypeptide encoded by the Lmt locus, variants or fragments thereof.
  • polypeptide refers to a polymer of amino acids and its equivalent and does not refer to a specific length of the product, thus, peptides, oligopeptides and proteins are included within the definition of a polypeptide. This term also does not refer to or exclude modifications of the polypeptide, for example, glycosylations, aceylations, phosphorylations and the like.
  • polypeptides containing one or more analogs of an amino acid including, for example, unnatural amino acids, etc.
  • polypeptides with substituted linkages as well as other modifications known in the art, both naturally and non-naturally occurring.
  • polypeptides will be at least about 40% similar to the natural LMT sequence, preferably in excess of 90% and more preferably at least about 95% similar.
  • proteins encoding by DNAs which hybridize under high or low stringency conditions to Lmt-encoding nucleic acids and closely related polypeptides or proteins retrieved by antisera to the LMT protein.
  • the mLMT polypeptide is shown in SEQ ID NO:2 and hLMT is shown in SEQ JD NO:24 and may be in isolated and/or purified form, free or substantially free of material with which it is naturally associated.
  • the polypeptide may, if produced by expression in a prokaryotic cell or produced synthetically, lack native post-translational processing, such as glycosylation.
  • the present invention is also directed to polypeptides which are sequence variants, alleles or derivatives of the LMT polypeptide.
  • Such polypeptides may have an amino acid sequence which differs from that set forth in SEQ ED NO:2 or SEQ ED NO:24 by one or more of addition, substitution, deletion or insertion of one or more amino acids. Conveniently, such polypeptides have LMT function but if not, they may nevertheless be useful in diagnostic or therapeutic assays.
  • Substitutional variants typically contain the exchange of one amino acid for another at one or more sites within the protein and may be designed to modulate one or more properties of the polypeptide such as stability against proteolytic cleavage without the loss of other functions or properties.
  • Amino acid substitutions may be made on the basis of similarity in polarity, charge, solubility, hydrophobicity, hydrophilicity and/or the amphipathic nature of the residues involved. Prefened substitutions are ones which are conservative, that is, one amino acid is replaced with one of similar shape and charge.
  • Conservative substitutions are well known in the art and typically include substitutions within the following groups: glycine, alanine; valine, isoleucine, leucine; aspartic acid, glutamic acid; asparagine, glutamine; serine, tlireonine; lysine, arginine; and tyrosine, phenylalanine.
  • Certain amino acids may be substituted for other amino acids in a protein structure without appreciable loss of interactive binding capacity with structures such as, for example, antigen- binding regions of antibodies or binding sites on substrate molecules or binding sites on proteins interacting with the LMT polypeptide. Since it is the interactive capacity and nature of a protein which defines that protein's biological functional activity, certain amino acid substitutions can be made in a protein sequence and its underlying DNA coding sequence and nevertheless obtain a protein with like properties. In making such changes, the hydropathic index of amino acids maybe considered. The importance of the hydrophobic amino acid index in conferring interactive biological function on a protein is generally understood in the art (Kyte and Doolittle, J. Mol. Biol. 157: 105-132, 1982).
  • hydrophilicity in conferring interactive biological function of a protein is generally understood in the art (U.S. Patent No. 4,554,101).
  • hydrophobic index or hydrophilicity in designing polypeptides is further discussed in U.S. Patent No. 5,691,198.
  • the length of the polypeptide sequences compared for homology will generally be at least about 16 amino acids, usually at least about 20 residues, more usually at least about 24 residues, typically at least about 28 residues and preferably more than about 35 residues.
  • the present invention further contemplates chemical analogs of the LMT polypeptide.
  • Analogs 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 analogs.
  • 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 4 ; 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
  • modifications of amino groups such as by reductive alkylation by reaction with an aldehyde followed by reduction with NaBH 4 ; 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); acy
  • 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 derivitization, for example, to a conesponding 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-chloromercuribenz ate, 4- chloromercuriphenylsulphonic acid, phenyhnercury 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 teframtromethane 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, ornithine, sarcosine, 4-amino-3-hydroxy-6-methylheptanoic acid, 2-thienyl alanine and/or D- isomers of amino acids.
  • a list of unnatural amino acid, contemplated herein is shown in Table
  • Non-conventional Code Non-conventional Code amino acid amino acid
  • D-N-methylcysteine Dnmcys N-(3 ,3 -diphenylpropyl)glycine Nbhe D-N-methylglutamine Dnmgln N-(3-guanidinopropyl) glycine Narg
  • peptides can be conformationally constrained by, for example, incorporation of C ⁇ and N ⁇ methylamino acids, introduction of double bonds between C ⁇ and C ⁇ atoms of amino acids and the formation of cyclic peptides or analogs 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.
  • peptide mimetic or “mimetic” is intended to refer to a substance which has the essential biological activity of the LMT polypeptide.
  • a peptide mimetic may be a peptide- containing molecule that mimics elements of protein secondary structure (Johnson et al, "Peptide Turn Mimetics” in Biotechnology and Pharmacy, Pezzuto et al, Eds., Chapman and Hall, New York, 1993).
  • the underlying rationale behind the use of peptide mimetics is that the peptide backbone of proteins exists chiefly to orient amino acid side chains in such a way as to facilitate molecular interactions such as those of antibody and antigen, enzyme and substrate or scaffolding proteins.
  • a peptide mimetic is designed to permit molecular interactions similar to the natural molecule.
  • a mimetic may not be a peptide at all, but it will retain the essential biological activity of natural LMT polypeptide.
  • the LMT polypeptide or fragment employed in such a test may either be free in solution, affixed to a solid support, or borne on a cell surface.
  • One method of drug screening utilizes eukaryotic or procaryotic host cells which are stably transformed with recombinant polynucleotides expressing the polypeptide or fragment, preferably in competitive binding assays. Such cells, either in viable or fixed form, can be used for standard binding assays.
  • One may measure, for example, the formation of complexes between a LMT polypeptide or fragment and the agent being tested, or examine the degree to which the formation of a complex between a LMT polypeptide or fragment and a known ligand is aided or interfered with by the agent being tested.
  • tumors have a propensity to develop in subjects in which at least one Lmt allele has been mutated or deleted so as to reduce or prevent its expression.
  • mutated is used in its most generic sense and includes methylation of all or part of the nucleotide sequences and in particular cytosine or guanine residues or other event which induces gene silencing.
  • the diagnostic and prognostic methods of the present invention detect or assess an abenation in the wild-type Lmt gene or locus, h addition, the method can be performed by detecting the wild-type Lmt locus and confirming a lack of a tumor or a predisposition to tumor development.
  • the term "abenation" in the Lmt gene or locus encompasses all forms of mutations including deletions, insertions, point mutations and substitutions in the coding and non-coding regions of Lmt. It also includes changes in methylation patterns of Lmt or of an allele of Lmt. Deletions may be of the entire gene or only a portion of the gene. Point mutations may result in stop codons, frameshift mutations or amino acid substitutions.
  • Somatic mutations are those which occur only in certain tissues, e.g. in the tumor tissue and are not inherited in the germline. Germline mutations can be found in any of a body's tissues and are inherited. A Lmt allele which is not deleted (e.g. that found on the sister chromosome to a chromosome canying a Lmt deletion) can be screened for other mutations such as insertions, small deletions, point mutations and changes in methylation pattern. It is considered in accordance with the present invention that many mutations found in tumor tissues are those leading to decreased expression of the Lmt gene. However, mutations leading to non-functional gene products would also likely lead to a cancerous state.
  • Point mutational events may occur in regulator regions, such as in the promoter of the gene or in an intron of the gene, leading to loss or diminution of expression of the mRNA. Point mutations may also abolish proper RNA processing leading to loss of expression of the Lmt gene product or a decrease in mRNA stability or translation efficiency. Point and other mutations may also affect proper RNA processing such as intron splicing.
  • Useful diagnostic techniques to detect abenations in Lmt include but are not limited to fluorescent in situ hybridization (FISH), direct DNA sequencing, PFGE analysis, Southern blot analysis, single-stranded coformational analysis (SSCA), Rnase protection assay, allele- specific oligonucleotide (ASO hybridization), dot blot analysis and PCR-SSCP (see below). Also useful is DNA microchip technology.
  • Predisposition to cancers can be ascertained by testing any tissue of a human for mutations of the Lmt gene.
  • leukemia of particular relevance and contemplated by the present invention is Acute Myeloid Leukemia (AML).
  • AML Acute Myeloid Leukemia
  • a person who has inherited a germline Lmt mutation would be prone to develop cancers. This can be determined by testing DNA from any tissue of a persons' body.
  • pre-natal diagnosis can be accomplished by testing fetal cells, placental cells or amniotic fluid for mutations of the Lmt gene. Alteration of a wild-type Lmt allele whether, for example, by point mutation or by deletion or by methylation, can be detected by any number of means.
  • tissue In order to detect the alteration of the wild-type Lmt gene in a tissue, it is helpful to isolate the tissue free from surrounding normal tissues.
  • Means for enriching a tissue preparation for tumor cells are known in the art.
  • the tissue may be isolated from paraffin or cryostat sections.
  • Cancer cells may also be separated from normal cells by flow cytometry. These techniques, as well as other techniques for separating tumor cells from normal cells, are well known in the art. If the tumor tissue is highly contaminated with normal cells, detection of mutations is more difficult.
  • SSCP single-stranded conformation polymorphism assay
  • CDGE clamped denaturing gel electrophoresis
  • HA heteroduplex analysis
  • CMC chemical mismatch cleavage
  • Other methods which might detect mutations in regulatory regions or which might comprise large deletions, duplications or insertions include the protein truncation assay or the asymmetric assay.
  • a rapid preliminary analysis to detect polymorphisms in DNA sequences can be performed by looking at a series of Southern blots of DNA cut with one or more restriction enzymes, preferably a large number of restriction enzymes. Each blot contains a series of normal individuals and a series of tumor cases. Southern blots displaying hybridizing fragments (differing in length from control DNA when probed with sequences near or including the Lmt locus) indicate a possible mutation. If restriction enzymes which produce very large restriction fragments are used, then pulsed field gel electrophoresis (PFGE) is employed.
  • PFGE pulsed field gel electrophoresis
  • Detection of point mutations may be accomplished by molecular cloning of the Lmt allele and sequencing that allele using techniques well known in the art.
  • the gene sequences can be amplified, using known techniques, directly from a genomic DNA preparation from the tumor tissue. The DNA sequence of the amplified sequences can then be determined.
  • primers are used which hybridize at their 3' ends to a particular Lmt mutation or to junctions of DNA caused by a deletion of Lmt. If the particular Lmt mutation is not present, an amplification product is not observed.
  • Amplification Refractory Mutation System (ARMS) can also be used, as disclosed in European Patent Publication No. 0 332 435 and in Newtown et al. (Nucl. Acids. Res. 17: 2503-2516, 1989). Insertions and deletions of genes can also be detected by cloning, sequencing and amplification.
  • restriction fragment length polymorphism (RFLP) probes for the gene or sunounding marker genes can be used to score alteration of an allele or an insertion in a polymorphic fragment. Such a method is particularly useful for screening relatives of an affected individual for the presence of the Lmt mutation found in that individual. Other techniques for detecting insertions and deletions as known in the art are used.
  • RFLP restriction fragment length polymorphism
  • DNA sequences of the Lmt gene which have been amplified by use of PCR or other amplification reactions may also be screened using allele-specific probes.
  • These probes are nucleic acid oligomers, each of which contains a region of the Lmt gene sequence harboring a known mutation. For example, one oligomer may be about 20-40 nucleotides in length, conesponding to a portion of the Lmt gene sequence.
  • PCR amplification products can be screened to identify the presence of a previously identified mutation in the Lmt gene.
  • Hybridization of allele-specific probes with amplified Lmt sequences can be performed, for example, on a nylon filter. Hybridization to a particular probe under stringent hybridization conditions indicates the presence of the same mutation in the tumor tissue as in the allele-specific probe.
  • Microchip technology is also applicable to the present invention.
  • thousands of distinct oligonucleotide or cDNA probes are built up in an array on a silicon chip or other solid support such as polymer films and glass slides.
  • Nucleic acid to be analyzed is labeled with a reporter molecule (e.g. fluorescent label) and hybridized to the probes on the chip. It is also possible to study nucleic acid-protein interactions using these nucleic acid microchips.
  • a reporter molecule e.g. fluorescent label
  • nucleic acid-protein interactions using these nucleic acid microchips.
  • the technique is described in a range of publications including Hacia et al.
  • the most definitive test for mutations in a candidate locus is to directly compare genomic Lmt sequences from cancer patients with those from a control population.
  • Mutations from cancer patients falling outside the coding region of Lmt can be detected by examining the non-coding regions such as introns and regulatory sequences near or within the Lmt gene including the promoter region.
  • An early indication that a mutation occurs in a non- coding region may come from Northern blot experiments which reveal the presence or absence of RNA molecules in cancer patients as compared to control individuals.
  • Alteration of Lmt mRNA expression can be detected by any of a number of techniques known in the art. These include Northern blot analysis, PCR amplification, RNase protection and microchip technology. Diminished mRNA expression indicates an alteration of the wild-type Lmt gene.
  • Alteration of wild-type Lmt genes can also be detected by screening for alteration of wild-type LMT proteins.
  • monoclonal antibodies immunoreactive with LMT can be used to screen a tissue. Lack of cognate antigen would indicate a LMT mutation.
  • Antibodies specific for products of mutant alleles could also be used to detect mutant Lmt gene product.
  • immunological assays can be done in any convenient format known in the art. These include Western blots, immunohistochemical assays and ELISA assays. Any means for detecting an altered LMT protein can be used to detect alteration of a wild-type Lmt gene.
  • the use of monoclonal antibodies in an immunoassay is particularly prefened 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 is derived by fusing an immortal cell line and lymphocytes sensitized against the immunogenic preparation (i.e. comprising LMT) or can be done by techniques which are well known to those who are skilled in the art. (See, for example, Douillard and Hoffman, Basic Facts about Hybridomas, in Compendium of Immunology Nol. II, ed. by Schwartz, 1981; Kohler and Milstein, Nature 256: 495-499, 1975; Kohler and Milstein, European Journal of Immunology 6: 511-519, 1976).
  • Another aspect of the present invention contemplates a method for detecting LMT in a biological sample from a subject, said method comprising contacting said biological sample with an antibody specific for LMT or its derivatives or homologs for a time and under conditions sufficient for an antibody-LMT complex to form, and then detecting said complex.
  • LMT may be accomplished in a number of ways such as by Western blotting, histochemistry and ELISA procedures.
  • a wide range of immunoassay techniques are available as can be seen by reference to U.S. Patent ⁇ os. 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 labeled antibody to a target. Loss of expression of LMT could be used as a diagnostic marker of Acute Myelogenous Leukaemia (AML).
  • AML Acute Myelogenous Leukaemia
  • the simplest method would be three- colour staining of permeabilized blood cells with a combination of fluorescently labeled anti- Lmt, anti-CD 19 and anti-CD45R antibodies.
  • This is based on 2 observations: first, the B- macrophage progenitors, which are distinctive in being CD19-positive and CD45R-negative, express abundant levels of Lmt mRNA, and second, the CD 19 population in AML bloods is suppressed in its expression of LMT).
  • Sandwich assays are among the most useful and commonly used assays and are favoured for use in the present invention.
  • an unlabeled antibody is immobilized on a solid substrate and the sample to be tested brought into contact with the bound molecule.
  • a second antibody specific to the antigen, labeled with a reporter molecule capable of producing a detectable signal is then added and incubated, allowing time sufficient for the formation of another complex of antibody-antigen-labeled antibody.
  • the antigen is LMT or a fragment thereof. Any unreacted material is washed away, and the presence of the antigen is determined by observation of a signal produced by the reporter molecule. The results may either be qualitative, by simple observation of the visible signal, or may be quantitated by comparing with a control ample containing known amounts of hapten. Variations on the forward assay include a simultaneous assay, in which both sample and labeled antibody are added simultaneously to the bound antibody. These techniques are well known to those skilled in the art, including any minor variations as will be readily apparent.
  • the sample is one which might contain LMT including cell extract or tissue biopsy. As LMT is an intracellular molecule, cell extracts and in particular nuclear extracts are prefened. The sample is, therefore, generally a biological sample comprising biological fluid.
  • a first antibody having specificity for the LMT 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 or 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 to the solid surface which is then 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 room temperature to about 37°C including 25°C) to allow binding of any subunit present in the antibody.
  • the antibody subunit solid phase is washed and dried and incubated with a second antibody specific for 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 the antigen.
  • 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 labeled with a reporter molecule. Depending on the amount of target and the strength of the reporter molecule signal, a bound target may be detectable by direct labelling with the antibody.
  • a second labeled 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.
  • reporter molecules in this type of assay are either enzymes, fluorophores or radionuclide containing molecules (i.e. radioisotopes) and chemiluminescent molecules.
  • an enzyme is conjugated to the second antibody, generally by means of glutaraldehyde or periodate.
  • glutaraldehyde or periodate As will be readily recognized, however, a wide variety of different conjugation techniques exist, which are readily available to the skilled artisan.
  • Commonly used enzymes include horseradish peroxidase, glucose oxidase, ⁇ - 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 conesponding enzyme, of a detectable color change. Examples of suitable enzymes include alkaline phosphatase and peroxidase.
  • fluorogenic substrates which yield a fluorescent product rather than the chromogenic substrates noted above.
  • the enzyme-labeled antibody is added to the first antibody hapten 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 hapten 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-labeled antibody When activated by illumination with light of a particular wavelength, the fluorochrome-labeled antibody absorbs the light energy, inducing a state to excitability in the molecule, followed by emission of the light at a characteristic color visually detectable with a light microscope.
  • the fluorescent labeled 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 the light of the appropriate wavelength, the fluorescence observed indicates the presence of the hapten of interest.
  • Immunofluorescene and EIA techniques are both very well established in the art and are particularly prefened for the present method. However, other reporter molecules, such as radioisotope, chemiluminescent or bioluminescent molecules, may also be employed.
  • Mutations which interfere with the function of the Lmt or LMT gene product are proposed to be involved in the pathogenesis of cancer.
  • the presence of an altered (or a mutant) Lmt gene which produces a protein having a loss of function, or altered function, or the lack of this protein directly increases the risk of cancer.
  • a biological sample is prepared and analyzed for a difference between the sequence of the allele being analyzed and the sequence of the wild-type allele.
  • Mutant Lmt alleles can be initially identified by any of the techniques described above. The mutant alleles are then sequenced to identify the specific mutation of the particular mutant allele. Alternatively, mutant alleles can be initially identified by identifying mutant (altered) proteins, using conventional techniques. The mutant alleles are then sequenced to identify the specific mutation for each allele.
  • the mutations, especially those which lead to an altered function of the protein are then used for the diagnostic and prognostic methods of the present invention.
  • an "isolated” or “substantially pure” nucleic acid is one which is substantially separated from other cellular components which naturally accompany a native sequence or protein, e.g. ribosomes, polymerases and many other genome sequences and proteins.
  • the term embraces a nucleic acid sequence or protein which has been removed from its naturally occurring environment and includes recombinant or cloned DNA isolates and chemically synthesized analogs or analogs biologically synthesized by heterologous systems.
  • the present invention provides methods of screening for drugs comprising contacting such an agent with a LMT polypeptide or fragment thereof and assaying (i) for the presence of a complex between the agent and the LMT polypeptide or fragment, or (ii) for the presence of a complex between the LMT polypeptide or fragment and a ligand, by methods well known in the art.
  • the LMT polypeptide or fragment is typically labeled. Free LMT polypeptide or fragment is separated from that present in a proteimprotein complex and the amount of free (i.e. uncomplexed) label is a measure of the binding of the agent being tested to LMT or its interference with or promotion of LMT:ligand binding, respectively.
  • Another technique for drug screening provides high throughput screening for compounds having suitable binding affinity to the LMT polypeptides and is described in detail in Geysen (International Patent Publication No. WO 84/03564). Briefly stated, large numbers of different small peptide test compounds are synthesized on a solid substrate, such as plastic pins or some other surface. The peptide test compounds are reacted with LMT polypeptide and washed. Bound LMT polypeptide is the detected by methods well known in the art.
  • Purified LMT can be coated directly onto plates for use in the aforementioned drug screening techniques.
  • non-neutralizing antibodies to the polypeptide can be used to capture antibodies to immobilize the LMT polypeptide on the solid phase.
  • the present invention also contemplates the use of competitive drug screening assays in which neutralizing antibodies capable of specifically binding the LMT polypeptide compete with a test compound for binding to the LMT polypeptide or fragments thereof. In this manner, the antibodies can be used to detect the presence of any peptide which shares one or more antigenic determinants of the LMT polypeptide.
  • the above screening methods are not limited to assays employing only LMT but are also applicable to studying LMT-protein complexes.
  • the effect of drugs on the activity of this complex, especially when either protein contains a mutation, is analyzed.
  • the substance may be investigated further. Furthermore, it may be manufactured and/or used in preparation, i.e. manufacture or formulation or a composition such as a medicament, pharmaceutical composition or drug. These may be administered to individuals in a method of treatment or prophylaxis.
  • the present invention extends, therefore, to a pharmaceutical composition, medicament, drug or other composition comprising LMT, a fragment thereof or a LMT mimetic or agonist, a method comprising administration of such a composition, a method comprising administration of such a composition to a patient, e.g. for treatment (which may include preventative treatment) of cancer, use of such a composition in the manufacture of a composition for administration, e.g. for treatment of cancer, and a method of making a pharmaceutical composition comprising admixing such a substance with a pharmaceutically acceptable excipient, vehicle or carrier, and optionally other ingredients.
  • a substance identified using as a modulator of polypeptide function may be peptide or non- peptide in nature.
  • Non-peptide "small molecules" are often prefened for many in vivo pharmaceutical uses. Accordingly, a mimetic or mimic of the substance (particularly if a peptide) may be designed for pharmaceutical use.
  • the designing of mimetics to a known pharmaceutically active compound is a known approach to the development of pharmaceuticals based on a "lead" compound. This might be desirable where the active compound is difficult or expensive to synthesize or where it is unsuitable for a particular method of admiiiistration, e.g. peptides are unsuitable active agents for oral compositions as they tend to he quickly degraded by proteases in the alimentary canal.
  • Mimetic design, synthesis and testing is generally used to avoid randomly screening large numbers of molecules for a target property.
  • a mimetic from a compound having a given target property There are several steps commonly taken in the design of a mimetic from a compound having a given target property.
  • These parts or residues constituting the active region of the compound are known as its "pharmacophore" . Once the pharmacophore has been found, its structure is modeled according to its physical properties, e.g. stereochemistry, bonding, size and/or charge, using data from a range of sources, e.g.
  • the three-dimensional structure of the ligand and its binding partner are modeled. This can be especially useful where the ligand and/or binding partner change conformation on binding, allowing the model to take account of this in the design of the mimetic.
  • a template molecule is then selected onto which chemical groups which mimic the pharmacophore can be grafted.
  • the template molecule and the chemical groups grafted onto it can conveniently be selected so that the mimetic is easy to synthesize, is likely to be pharmacologically acceptable, and does not degrade in vivo, while retaining the biological activity of the lead compound.
  • the mimetic is peptide-based
  • further stability can be achieved by cyclizing the peptide, increasing its rigidity.
  • the mimetic or mimetics found by this approach can then be screened to see whether they have the target property, or to what extent they exhibit it. Further optimization or modification can then be carried out to arrive at one or more final mimetics for in vivo or clinical testing.
  • the goal of rational drug design is to produce structural analogs of biologically active polypeptides of interest or of small molecules with which they interact (e.g. agonists, antagonists, inhibitors or enhancers) in order to fashion drugs which are, for example, more active or stable forms of the polypeptide, or which, e.g. enhance or interfere with the function of a polypeptide in vivo. See, e.g. Hodgson (Bio/Technology 9: 19-21, 1991).
  • a protein of interest e.g. LMT
  • LMT three-dimensional structure of a protein of interest
  • peptides e.g. LMT
  • Wells, Methods Enzymol 202: 2699-2705, 1991 an amino acid residue is replaced by Ala and its effect on the peptide' s activity is determined.
  • Each of the amino acid residues of the peptide is analyzed in this manner to determine the important regions of the peptide.
  • drugs which have, e.g. improved LMT activity or stability or which act as enhancers, inhibitors, agonists, antagonists, etc. of LMT activity.
  • sufficient amounts of the LMT polypeptide may be made available to perform such analytical studies as x-ray crystallography.
  • the knowledge of the LMT protein sequence provided herein will guide those employing computer modeling techniques in place of, or in addition to x-ray crystallography.
  • a method is also provided of supplying wild-type Lmt function to a cell which carries a mutant Lmt allele. Supplying such a function should suppress neoplastic growth of the recipient cells.
  • the wild-type Lmt gene or a part of the gene may be introduced into the cell in a vector such that the gene remains extrachromosomal. In such a situation, the gene will be expressed by the cell from the extrachromosomal location. If a gene portion is introduced and expressed in a cell carrying a mutant Lmt allele, the gene portion should encode a part of the LMT protein which is require for non-neoplastic growth of the cell.
  • More prefened is the situation where the wild-type Lmt gene or a part thereof is introduced into the mutant cell in such a way that it recombines with the endogenous mutant Lmt gene present in the cell. Such recombination requires a double recombination event which results in the conection of the Lmt gene mutation.
  • Vectors for introduction of genes both for recombination and for extrachromosomal maintenance are known in the art and any suitable vector may be used.
  • Methods for introducing DNA into cells such as electroporation calcium phosphate co-precipitation and viral transduction are known in the art and the choice of method is within the competence of the practitioner.
  • Cells transformed with the wild-type Lmt gene can be used as model systems to study cancer remission and drug treatments which promote such remission.
  • the Lmt gene or fragment may be employed in gene therapy methods in order to increase the amount of the expression products of such genes in cancer cells.
  • Such gene therapy is particularly appropriate for use in both cancerous and pre-cancerous cells, in which the level of LMT polypeptide is absent or diminished compared to normal cells. It may also be useful to increase the level of expression of a given Lmt gene even in those tumor cells in which the mutant gene is expressed at a "normal" level, but the gene product is not fully functional.
  • Gene therapy would be carried out according to generally accepted methods, for example, as described by Friedman (In: Therapy for Genetic Disease, T. Friedman, Ed., Oxford University Press, pp. 105-121, 1991) or Culver (Gene Therapy: A Primer for Physicians, 2 nd Ed., Mary Ann Liebert, 1996).
  • Cells from a patient's tumor would be first analyzed by the diagnostic methods described above, to ascertain the production of LMT polypeptide in the tumor cells.
  • a virus or plasmid vector, containing a copy of the Lmt gene linked to expression control elements is prepared.
  • the vector may be capable of replicating inside the tumor cells. Suitable vectors are known, such as disclosed in U.S. Patent No. 5,252,479, International Patent Publication No.
  • WO 93/07282 and U.S. Patent No. 5,691,198 The vector is then injected into the patient, either locally at the site of the tumor or systemically (in order to reach any tumor cells that may have metastasized to other sites). If the transfected gene is not permanently incorporated into the genome of each of the targeted tumor cells, the treatment may have to be repeated periodically.
  • Gene transfer systems known in the art may be useful in the practice of the gene therapy methods of the present invention. These include viral and non- viral transfer methods.
  • viruses have been used as gene transfer vectors or as the basis for preparing gene transfer vectors, including papovaviruses (e.g. SV40, Madzak et al, J. Gen. Virol. 73: 1533- 1536, 1992), adenovirus (Berkner, Curr. Top. Microbiol. Immunol. 158: 39-66, 1992; Berkner et al, BioTechniques 6; 616-629, 1988; Gorziglia and Kapikian, J. Virol. 66: 4407-4412, 1992; Quantin et al, Proc. Natl.
  • Non-viral gene transfer methods are known in the art such as chemical techniques including calcium phosphate co-precipitation, mechanical techniques, for example, microinjection, membrane fusion-mediated transfer via liposomes and direct DNA uptake and receptor- mediated DNA transfer.
  • Viral-mediated gene transfer can be combined with direct in vivo gene transfer using liposome delivery, allowing one to direct the viralvectors to the tumor cells and not into the sunounding non-dividing cells.
  • the retroviral vector producer cell line can be injected into tumors. Injection of producer cells would then provide a continuous source of vector particles.
  • Lu an approach which combines biological and physical gene transfer methods, plasmid DNA of any size is combined with a polylysine-conjugated antibody specific to the adenovirus hexon protein and the resulting complex is bound to an adenovirus vector. The trimolecular complex is then used to infect cells.
  • the adenovirus vector permits efficient binding, internalization and degradation of the endosome before the coupled DNA is damaged.
  • U.S. Patent No. 5,691,198 see U.S. Patent No. 5,691,198.
  • Liposome/DNA complexes have been shown to be capable of mediating direct in vivo gene transfer. While in standard liposome preparations the gene transfer process is non-specific, localized in vivo uptake and expression have been reported in tumor deposits, for example, following direct in situ administration (Nabel, [1992; supra]).
  • Expression vectors in the context of gene therapy are meant to include those constructs containing sequences sufficient to express a polynucleotide that has been cloned therein. In viral expression vectors, the construct contains viral sequences sufficient to support packaging of the construct. If the polynucleotide encodes LMT, expression will produce LMT.
  • the polynucleotide encodes a sense or antisense polynucleotide or a ribozyme or DNAzyme
  • expression will produce the sense or antisense polynucleotide or ribozyme or DNAzyme.
  • expression does not require that a protein product be synthesized.
  • the vector also contains a promoter functional in eukaryotic cells.
  • the cloned polynucleotide sequence is under control of this promoter. Suitable eukaryotic promoters include those described above.
  • the expression vector may also include sequences, such as selectable markers and other sequences described herein.
  • Receptor- mediated gene transfer is accomplished by the conjugation of DNA (usually in the form of covalently closed supercoiled plasmid) to a protein ligand via polylysine.
  • Ligands are chosen on the basis of the presence of the conesponding ligand receptors on the cell surface of the target cell/tissue type.
  • These ligand-DNA conjugates can be injected directly into the blood if desired and are directed to the target tissue where receptor binding and internalization of the DNA-protein complex occurs.
  • co-infection with adenovirus can be included to disrupt endosome function.
  • the therapy is as follows: patients who carry an altered Lmt allele are treated with a gene delivery vehicle such that some or all of their precursor cells receive at least one additional copy of a functional normal Lmt allele. In this step, the treated individuals have reduced risk of cancer to the extent that the effect of the susceptible allele has been countered by the presence of the normal allele.
  • Peptides which have LMT activity can be supplied to cells which cany mutant or missing Lmt alleles.
  • the sequence of the LMT protein is disclosed (SEQ ED NO:2 or SEQ ED NO:24). Protein can be produced by expression of the cDNA sequence in bacteria, for example, using known expression vectors. Alternatively, LMT polypeptide can be extracted from LMT- producing mammalian cells.
  • the techniques of synthetic chemistry can be employed to synthesize LMT protein. Any of such techniques can provide the preparation of the present invention which comprises the LMT protein. The preparation is substantially free of other proteins. This is most readily accomplished by synthesis in a microorganism or in vitro.
  • Active LMT molecules can be introduced into cells by microinjection or by use of liposomes, for example. Alternatively, some active molecules may be taken up by cells, actively or by diffusion. Extracellular application of the LMT product may be sufficient to affect tumor growth. Supply of molecules with LMT activity should lead to partial reversal of the neoplastic state. Other molecules with LMT activity (for example, peptides, drugs or organic compounds) may also be used to effect such a reversal. Modified polypeptides having substantially similar function are also used for peptide therapy.
  • cells and animals which cany a mutant Lmt allele or where one or both alleles are deleted can be used as model systems to study.
  • Mice, rats, rabbits, guinea pigs, hamsters and zebrafish are particularly useful as model systems.
  • the cells may be isolated from individuals with Lmt mutations, either somatic or germline. Alternatively, the cell line can be engineered to carry the mutation in the Lmt allele, as described above. After a test substance is applied to the cells, the neoplastically transformed phenotype of the cell is determined.
  • neoplastically transformed cells can be assessed, including anchorage-independent growth, tumorigenicity in nude mice, invasiveness of cells and growth factor dependence. Assays for each of these traits are known in the art. Animals for testing therapeutic agents can be selected after mutagenesis of whole animals or after treatment of germline cells or zygotes. Such treatments include insertion of mutant Lmt alleles, usually from a second animal species, as well as insertion of disrupted homologous genes. Alternatively, to endogenous Lmt gene of the animals may be disrupted by insertion or deletion mutation or other genetic alterations using conventional techniques. After test substances have been administered to the animals, the growth of tumors must be assessed.
  • test substance prevents or suppresses the growth of tumors
  • test substance is a candidate therapeutic agent for the treatment of the cancers identified herein.
  • animal models provide an extremely important testing vehicle for potential therapeutic products.
  • heterologous Lmt genes can be introduced into an animal.
  • non- murine Lmt genes e.g. hLmt
  • the present invention provides, therefore, an animal model useful for screening for agents capable of ameliorating the effects of Lmt or LMT.
  • the animal model produces low amounts of LMT.
  • Such an animal would have a predisposition for a range of diseases including cancers and tumors.
  • the animal model is useful for screening for agents which ameliorate such conditions.
  • Another aspect of the present invention provides a genetically modified animal wherein said animal produces low amounts of LMT relative to a non-genetically modified animal of the same species.
  • Reference to "low amounts” includes zero amounts or up to about 10% lower than normalized amounts.
  • the genetically modified animal is a mouse, rat, guinea pig, rabbit, pig, sheep, goat or zebrafish. More preferably, the genetically modified animal is a mouse or rat.
  • the animal model contemplated by the present invention comprises, therefore, an animal which is substantially incapable of producing LMT. Generally, but not exclusively, such an animal is refened to as a homozygous or heterozygous LMT-knockout animal.
  • the animal models of the present invention may be in the form of the animals including fish or may be, for example, in the form of embryos for transplantation. The embryos are preferably maintained in a frozen state and may optionally be sold with instructions for use.
  • the genetically modified animals may also produce larger amounts of LMT.
  • over expression of normal Lmt or mutant Lmt may produce dominant negative effects and may become useful disease models.
  • Another aspect of the present invention is directed to a genetically modified animal over-expressing genetic sequences encoding Lmt.
  • a genetically modified animal includes a transgenic animal, or a "knock-out” or “knock-in” animal. Furthermore, co-suppression may be used to induce post-transcriptional gene silencing. Co-suppression includes induction of RNAi.
  • Yet another aspect of the present invention provides a targeting vector useful for inactivating a gene encoding Lmt, said targeting vector comprising two segments of genetic material encoding said Lmt flanking a positive selectable marker wherein when said targeting vector is transfected into embryonic stem (ES) cells and the marker selected, an ES cell is generated in which the gene encoding said Lmt is inactivated by homologous recombination.
  • ES embryonic stem
  • the ES cells are from mice, rats, guinea pigs, pigs, sheep, goats or zebrafish.
  • Still yet another aspect of the present invention is directed to the use of a targeting vector as defined above in the manufacture of a genetically modified animal substantially incapable of producing LMT.
  • One particularly useful animal model employs dominant negative Lmt mutant genes expressed in murine or other animals.
  • heteroduplexes form between the wild-type and dominant negative LMT proteins resulting in inactivation of the wild-type LMT. As a result, the murine animal becomes predisposed to cancer development.
  • Lmt alleles are screened for mutations either directly or after cloning the alleles.
  • the alleles are tested for the presence of nucleic acid sequence differences from the normal allele using any suitable technique, including but not limited to, one of the methods herein before described, i.e.
  • FISH fluorescent in situ hybridization
  • direct DNA sequencing PFGE analysis
  • Southern blot analysis single-stranded conformation analysis (SSCP)
  • linkage analysis RNase protection assay
  • ASO allele specific oligonucleotide
  • dot blot analysis PCR-SSCP analys, or DNA/RNA microchip technology.
  • Two-hybrid screening is particularly useful in identifying other members of a biochemical or genetic pathway associated with Lmt.
  • Two-hybrid screening conveniently uses Saccharomyces cerevisiae and Saccharomyces pombe. LMT interactions and screens for inhibitors can be carried out using the yeast two-hybrid system, which takes advantage of transcriptional factors that are composed of two physically separable, functional domains (Phizicky and Fields, 1994). The most commonly used is the yeast GAL4 transcriptional activator consisting of a DNA binding domain and a transcriptional activation domain. Two different cloning vectors are used to generate separate fusions of the GAL4 domains to genes encoding potential binding proteins.
  • the fusion proteins are co-expressed, targeted to the nucleus and if interactions occur, activation of a reporter gene (e.g. lacZ) produces a detectable phenotype.
  • a reporter gene e.g. lacZ
  • S. cerevisiae is co-transformed with a library or vector expressing a cDNA GAL4 activation domain fusion and a vector expressing a LMT-GAL4 binding domain fusion.
  • lacZ is used as the reporter gene, co-expression of the fusion proteins will produce a blue colour. Small molecules or other candidate compounds which interact with LMT will result in loss of colour of the cells.
  • This system can be used to screen for small molecules that inhibit the LMT interaction and, hence, protect the yeast against cell death and to determine the residues in LMT which are involved in their interaction.
  • yeast two-hybrid systems as disclosed by Munder et al. (Appl. Microbiol. Biotechnol. 52(3): 311-320, 1999) and Young et al, Nat. Biotechnol. 16(10): 946-950, 1998). Molecules thus identified by this system are then re-tested in animal cells.
  • the LMT polypeptides, antibodies, peptides, chemical analogs or mimetics and nucleic acids of the present invention can be formulated in pharmaceutic compositions which are prepared according to conventional pharmaceutical compounding techniques. See, for example, Remington's Pharmaceutical Sciences, 18 th Ed. (1990, Mack Publishing, Company, Easton, PA, U.S.A.).
  • the composition may contain the active agent or pharmaceutically acceptable salts of the active agent.
  • These compositions may comprise, in addition to one of the active substances, a pharmaceutically acceptable excipient, carrier, buffer, stabilizer or other materials well known in the art. Such materials should be non-toxic and should not interfere with the efficacy of the active ingredient.
  • the carrier may take a wide variety of forms depending on the form of preparation desired for administration, e.g. intravenous, oral, intrathecal, epineural or parenteral.
  • the compounds can be formulated into solid or liquid preparations such as capsules, pills, tablets, lozenges, Lmt, powders, suspensions or emulsions.
  • any of the usual pharmaceutical media may be employed, such as, for example, water, glycols, oils, alcohols, flavoring agents, preservatives, coloring agents, suspending agents, and the like in the case of oral liquid preparations (such as, for example, suspensions, elixirs and solutions); or carriers such as starches, sugars, diluents, granulating agents, lubricants, binders, disintegrating agents and the like in the case of oral solid preparations (such as, for example, powders, capsules and tablets).
  • tablets and capsules represent the most advantageous oral dosage unit form, in which case solid pharmaceutical carriers are obviously employed. If desired, tablets may be sugar-coated or enteric-coated by standard techniques.
  • the active agent can be encapsulated to make it stable to passage through the gastrointestinal tract while at the same time allowing for passage across the blood brain barrier. See for example, International Patent Publication No. WO 96/11698.
  • the compound may dissolved in a pharmaceutical carrier and administered as either a solution of a suspension.
  • suitable carriers are water, saline, dextrose solutions, fructose solutions, ethanol, or oils of animal, vegetative or synthetic origin.
  • the carrier may also contain other ingredients, for example, preservatives, suspending agents, solubilizing agents, buffers and the like.
  • the compounds When the compounds are being admimstered intrathecally, they may also be dissolved in cerebrospinal fluid.
  • the active agent is preferably administered in a therapeutically effective amount.
  • the actual amount administered and the rate and time-course of administration will depend on the nature and severity of the condition being treated. Prescription of treatment, e.g. decisions on dosage, timing, etc. is within the responsibility of general practitioners or specialists and typically takes account of the disorder to be treated, the condition of the individual patient, the site of delivery, the method of administration and other factors known to practitioners. Examples of techniques and protocols can be found in Remington's Pharmaceutical Sciences.
  • targeting therapies may be used to deliver the active agent more specifically to certain types of cell, by the use of targeting systems such as antibodies or cell specific ligands.
  • Targeting may be desirable for a variety of reasons, e.g. if the agent is unacceptably toxic or if it would otherwise require too high a dosage or if it would not otherwise be able to enter the target cells.
  • they could be produced in the target cell, e.g. in a viral vector such as described above or in a cell based delivery system such as described in U.S. Patent No. 5,550,050 and Intemational Patent Publication Nos.
  • the vector could be targeted to the target cells.
  • the cell based delivery system is designed to be implanted in a patient's body at the desired target site and contains a coding sequence for the target agent.
  • the agent could be administered in a precursor form for conversion to the active form by an activating agent produced in, or targeted to, the cells to be treated. See, for example, European Patent Application No. 0 425 731 A and International Patent Publication No. WO 90/07936.
  • Myeloid leukemia was induced in CBA X SJL/J male mice (Animal Resource Centre, Perth) by treatment with hydrocortisone and X-inadiation (Alexander et al, Leukemia 9: 2009-2015, 1995).
  • Loss of heterozygosity (LOH) at microsatellite markers was used to identify 58 deleted tumors, and to map a commonly deleted region ( Figure 1). The same region was shown to be lost in 3 in v/tro-generated deletions in the clonal hemopoietic tumor cell line RB22.2 (Alexander et al, Genes Chrom Cancer 5: 286-298, 1992).
  • Figure 1 is a diagrammatic representation of YACs and BACs showing particular markers identified over the deleted regions.
  • EXAMPLE 2 Identification of translated nucleotide sequences within cdr
  • cdr transcripts were assessed by RT-PCR for expression in cell lines derived from the deleted tumors and the deleted RB22.2 derivatives, compared with normal spleen and bone manow, the non-deleted RB22.2 derivatives and non-deleted CBA X SJL/J hemopoietic cell lines. Only one transcript, Lmt, showed both expression in the normal hemopoietic tissues and non-deleted cell lines, and reduced expression in the deleted cell lines. This is demonstrated in the upper panel of Figure 2A. Compare this with the lower panel, which shows the uniform expression of another gene from the cdr, MADD.
  • the Celera promo program predicted a Lmt gene in the Celera mouse sequence consisting of 9 exons, spread over 140 kb.
  • RACE experiments confirmed that the transcript terminated at the 3' end of the last of these exons, and identified an extra 5' exon that was located in the Celera mouse genome sequence approximately 20 kb upstream of the initial exon.
  • the inventors concluded that the Lmt gene is composed of 10 exons, spread over 160 kb.
  • the coding sequence for mLmt is shown in SEQ ID NO:l with the conesponding amino acid sequence for the polypeptide product of mLmt, i.e. LMT, shown in SEQ ED NO:2.
  • Introns of mLmt are shown in SEQ ID NOs: 6 through 13 and exons are shown in SEQ ED NOs:14 through 22.
  • the coding region of the Lmt gene is highly conserved among mammalian species.
  • the amino acid sequence of the mouse ortholog is 93% identical and 97% conserved, compared with the human protein ( Figure 3A).
  • the only non-orthologous homology found is for a central domain of 105 amino acids (128-233, Figure 3B), which is weakly similar (17% identical, 45%) conserved) to the N-terminal protein-protein interaction domain of the STAT family (Xu et al, Science 273: 794-797, 1996), and part of which also aligns (46% identical, 60% conserved) with a region of unknown function in unc86, the C. elegans transcription factor (Finney et al, Cell 55: 757-769, 1988), and with an EST from the ascidian Ciona intestinalis (47% identical, 63% similar).
  • the human Lmt coding region was amplified by PCR using the primers shown in Methods below, which were identified in the orthologous human EST, Genbank accession no 13129129 (see Figure 3), and cDNA made from 293T cells (Graham et al, J. Gen. Virol. 36: 59-72, 1977).
  • the mouse cDNA sequence was inserted into the expression vector pEGFP, and transiently transfected into various cell lines by lipofection.
  • RNA and DNA will be extracted.
  • RNA will be analyzed by Northern blot, using the human PCR product as probe.
  • Polymorphisms will be identified from the Celera genome database, and if necessary by PCR amplification across repetitive regions, as the inventors have done for the mouse Lmt region and used to search for loss of heterozygosity.
  • nucleotide sequence of the open reading frame of hLmt is shown in SEQ ED NO:23 with the conesponding amino acid sequence given is given in SEQ ED NO:24.
  • cDNA from the tumors and cell lines will be synthesized and amplified by PCR for sequencing. Cells will be treated with 5-aza-2-deoxycytidine to test for methylation.
  • the combination of these approaches will reveal any deletions, mutations and methylation- mediated silencing of the hLmt gene.
  • deletion of the human chromosomal region around Lmt (1 lpl 1-12) has been demonstrated in both leukemia and melanoma (Berger et al, Genes Chrom. Cancer 3: 332-337, 1991; Goldberg et al, Am. J. Hum. Genet. 67: 417-431, 2000)
  • the inventors predict that at least one of these mechanisms will diminish hLmt expression, most likely in leukemias and melanomas.
  • the human kidney fibroblast cell line 293T was obtained from The Walter and Eliza Hall Institute of Medical Research, Australia). 5-aza-2deoxycytidine (Sigma) was titrated for maximum dose without evidence of toxicity over 48 hr, which was determined to be 1 ⁇ M.
  • Plasmid constructs were transiently transfected into cells using Lipofectamine 2000 (Invitrogen), in RPMI, 5% w/v FCS according to the manufacturer's instructions. After a 48 hr expression period, fluorescent cells were sorted by a FACS II flow cytometer, plated at 700 cells/well in 96-well plates, and re- incubated at 37°C, 5% CO 2 . Six replicate wells from each sample were assayed per day for 3 days by adding 20 ⁇ l of MTS/PMS (Promega), and measuring absorbance at 490 nm after 4 hr further incubation.
  • YAC and BAC libraries and sublibraries YACs from the WI/MIT820 library and BACs from the RPCI-23 library were obtained from Research Genetics and the Australian Genome Research Facility, respectively. Microsatellite markers were screened using primers from Research Genetics. Gridded BAC filters (BACPAC Resources, Roswell Park Cancer Institute) were screened by hybridization with ⁇ - P-labelled pnmers and the identities of selected BACs were confirmed by PCR. YACs were screened by acrylamide electrophoresis of PCR products generated using the microsatellite primers. YAC-end sequences were obtained by a vector- hexamer PCR technique as described (Herring et al, Genome Res.
  • BAC sizes were estimated from pulse-field gel electrophoresis, compared with mid-range PFG markers I (New England Biolabs). Sub-libraries of HaeUI and Rsal digested BACs were constructed in pBluescriptII(KS+), and screened for CA repeats using a (CA) 15 oligomer. Positive clones were confirmed by PCR, and sequences were used to eliminate redundancies and known microsatellites and to design new primers.
  • cDNA was synthesized from bone marrow RNA using, as primer for 5' RACE: 5' GTA TAA AGC TGA ATT CCC GGA AGA 3' [SEQ ED NO:45], or for 3' RACE: WC245 (5' GAC TCG AGT CGA CAT CGA TTT TTT TTT TTT TTT TT 3' [SEQ JD NO:46]).
  • the cDNA was purified using a QIAGEN QIAquick spin column according to the manufacturer's instructions.
  • the 5' cDNA was tailed with dGTP using di-deoxy terminal transferase, and re-purified.
  • RNA samples 15 ⁇ g per sample of total RNA were electrophoresed through agarose, blotted onto a nylon membrane, and probed with DNA fragments labelled with 32 P by random primer extension, as described (Alexander et al. [1995; supra]).
  • Purified plasmids or PCR products were sequenced using an ABI BigDye terminator Sequencing Kit (Perkin-Elmer) and an ABI 377 Sequenator and analyzed using Sequencing Analysis and Base Caller PPC (Perkin Elmer).
  • a number of animal models are used to analyze Lmt and to screen for potential anti-cancer agents.
  • a nucleic acid molecule comprising a nucleotide sequence encoding a dominant negative LMT protein is introduced into a murine animal.
  • so-called "knockout" mice are produced, from embryonic cells in which the endogenous Lmt gene has been disabled by insertion of exogenous DNA.
  • the transgenic or knockout murine animal is predisposed to cancer. The pathogenesis of cancer may then be studied and/or the animal screened with potential anti-cancer agents.
  • a homologous or heterologous Lmt gene is overexpressed in a murine animal.
  • Such animals may either have a reduced capacity to form tumors or an enhanced capacity to form tumors.
  • EXAMPLE 11 Morpholino antisense oligonucleotides
  • Antisense oligonucleotides are generated using morpholinos, which are oligonucleotides composed of morpholine nucleotide derivatives and phosphorodiamidate linkages (Summerton Weller, 1997, supra). Such compounds are injected into zebrafish embryos, where they are taken up efficiently by cells, and the effect of interference with mRNA is seen within hours. It is predicted that such experiments using antisense Lmt morpholinos would result in disruption of normal zebrafish haemopoiesis, and possibly excessive growth of myeloid cells.
  • a zebrafish cDNA library is probed with the partial cDNA sequence Genbank AI883859, which has been identified as being orthologous to Lmt. Identified cDNAs are sequenced and compared to mouse and human Lmt. The longest cDNA fragments are then extended by 5' and 3' RACE to generate a full-length cDNA sequence.
  • the cDNA sequence form zebrafish is used to generate antisense molecules and knock-out fish as animal models.
  • cDNA dot blot (Clontech) analysis is used to screen for the presence or absence of Lmt expression in various human subjects.
  • Whole blood is collected from subjects divided on the basis of being one of "normal", having AML, having CML, having non-Hodgkin's lymphoma, having Hodgkin's lymphoma and having von Willebrand's disease.
  • the whole blood is subjected to FACS fractionation to fractionate the blood into polymorph, monocyte, CD3 (T- cell), CD19 (mostly B-cell) and CD14 (myeloid) cell populations.
  • a total unfractionated pool is also employed.
  • Arrays of cDNAs from each source are used based on individual subjects versus fractionated whole blood.
  • the cDNA anays are subjected to hybridization analysis with a hLmt probe.
  • the CD19 population mainly B-cells
  • the CD19 population revealed significantly weaker hybridization signals for hLmt.
  • the nature of this type of analysis is likely to be an under-estimation of the reduction in expression.

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Abstract

La présente invention relève d'une manière générale du domaine du traitement du cancer et du dépistage du cancer et concerne des agents utilisés à cet effet. De manière plus spécifique, la présente invention concerne un nouveau gène suppresseur de tumeur, un produit d'expression comprenant un polypeptide ou une protéine codée par ce produit d'expression et des sondes et amorces nucléotidiques et des fragments peptidiques caractéristiques du gène suppresseur de tumeur ou de son produit d'expression ou des mutants ou dérivés de celui-ci. Cette invention concerne également des molécules générées par chimie combinatoire ou synthèse chimique rationnelle ou criblage suivant identifié de bibliothèques chimiques ou sources naturelles qui augmentent ou facilitent l'activité ou la fonction du gène supresseur de tumeur ou de son produit d'expression et en particulier dans des cellules qui portent une délétion ou autre mutation comprenant un événement de silençage génique dans un ou les deux allèles du gène suppresseur de tumeur. Cette invention concerne également des agents de diagnostic permettant de détecter la présence ou l'absence du gène suppresseur de tumeur expressible ou la présence ou l'absence d'un produit d'expression du gène suppresseur de tumeur utilisé pour déterminer la probabilité du développement d'une tumeur chez un animal vertébré et en particulier chez un être humain. Cette invention concerne également des animaux génétiquement modifiés porteurs de cellules dans lesquelles un ou les deux allèles du gène suppresseur de tumeur sont supprimés ou mutés ou pour lesquels l'expression du gène est silencée.
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